RESUMO
Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.
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Rim , Multiômica , Humanos , Diferenciação Celular/genética , Células Cultivadas , Organoides/metabolismo , Análise de Célula ÚnicaRESUMO
In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase (PARP) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI.NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of siTNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.
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Injúria Renal Aguda , Apoptose , Células Epiteliais , Túbulos Renais Proximais , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Animais , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/genética , Transdução de Sinais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Linhagem Celular , Inflamação/metabolismo , Inflamação/patologia , MasculinoRESUMO
SIGNIFICANCE STATEMENT: HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development. BACKGROUND: The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated. METHODS: We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation. RESULTS: Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation. CONCLUSIONS: This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.
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Redes Reguladoras de Genes , Rim , Humanos , Camundongos , Animais , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Regulação da Expressão Gênica , Organoides/metabolismo , Fator 4 Nuclear de Hepatócito/genéticaRESUMO
'Kitahonami' is a soft red winter wheat (Triticum aestivum L.) cultivar that has high yield, good agronomic performance and good quality characteristics. It currently accounts for 73% of the wheat cultivation area of Hokkaido the northern island in Japan and 42% of Japan's overall wheat cultivation. However, this cultivar is susceptible to Wheat yellow mosaic virus (WYMV). WYMV has become widespread recently, with serious virus damage reported in Tokachi and Ohotsuku districts, which are the main wheat production areas in Hokkaido. Here, we report a new wheat breeding line 'Kitami-94', which was developed over four years by repeated backcrossing with 'Kitahonami' using DNA markers for WYMV resistance linked to the Qym1 and Qym2 from 'Madsen'. Basic maps of Qym1 and Qym2 were created and used to confirm that 'Kitami-94' reliably carried the two resistance genes. 'Kitami-94' demonstrated WYMV resistance, and had agronomic traits and quality equivalent to 'Kitahonami' except for higher polyphenol oxidase activity and lower thousand grain weight. 'Kitami-94' may be useful for elucidating the mechanism of WYMV resistance in the background of 'Kitahonami', and for developing new cultivars.
RESUMO
BACKGROUND: Previous research has elucidated the signals required to induce nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling the generation of kidney organoids. However, selectively controlling differentiation of NPCs to podocytes has been a challenge. METHODS: We investigated the effects of various growth factors in cultured mouse embryonic NPCs during three distinct steps of nephron patterning: from NPC to pretubular aggregate, from the latter to epithelial renal vesicle (RV), and from RV to podocyte. We then applied the findings to human PSC-derived NPCs to establish a method for selective induction of human podocytes. RESULTS: Mouse NPC differentiation experiments revealed that phase-specific manipulation of Wnt and Tgf-ß signaling is critical for podocyte differentiation. First, optimal timing and intensity of Wnt signaling were essential for mesenchymal-to-epithelial transition and podocyte differentiation. Then, inhibition of Tgf-ß signaling supported domination of the RV proximal domain. Inhibition of Tgf-ß signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resultant protocol enabled successful induction of human podocytes from PSCs with >90% purity. The induced podocytes exhibited global gene expression signatures comparable to those of adult human podocytes, had podocyte morphologic features (including foot process-like and slit diaphragm-like structures), and showed functional responsiveness to drug-induced injury. CONCLUSIONS: Elucidation of signals that induce podocytes during the nephron-patterning process enabled us to establish a highly efficient method for selective induction of human podocytes from PSCs. These PSC-derived podocytes show molecular, morphologic, and functional characteristics of podocytes, and offer a new resource for disease modeling and nephrotoxicity testing.
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Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/transplante , Podócitos/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Camundongos , Camundongos Transgênicos , Néfrons/citologia , Organoides/metabolismo , Células-Tronco Pluripotentes/citologia , Transdução de SinaisRESUMO
The glomerular podocyte is one of the major targets of kidney research. Recent establishment of kidney organoids from pluripotent stem cells has enabled the detailed analysis of human podocytes in both development and disease. The podocytes in organoids express slit diaphragm-related genes and proteins and exhibit characteristic morphology, especially upon experimental transplantation. Organoid technology is now used to reproduce hereditary podocyte diseases, and selective podocyte induction methods have also been reported. Moreover, single-cell RNA-sequencing of human fetal and adult kidneys has revealed the detailed molecular features of this cell lineage, as well as serving as references for kidney organoids in which podocytes are still immature. Here, we discuss the recent progress and limitations of podocyte research from the viewpoint of developmental biology and kidney organoids.
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Podócitos , Pesquisa com Células-Tronco , Animais , Humanos , Células-Tronco Pluripotentes Induzidas , OrganoidesRESUMO
Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.
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Células-Tronco Pluripotentes Induzidas/citologia , Glomérulos Renais/irrigação sanguínea , Podócitos/fisiologia , Animais , Transplante de Células , Células Cultivadas , Humanos , Camundongos , Podócitos/ultraestruturaRESUMO
KEY MESSAGE: Wheat yellow mosaic virus resistance of Madsen is governed by two complementary QTLs, Qym1 and Qym2 , located on chromosome arms 2DL and 3BS. Wheat yellow mosaic, caused by Wheat yellow mosaic virus (WYMV), is one of the most serious wheat diseases in East Asia. In this study, recombinant inbred lines (RILs, F9) from a cross between cultivars Madsen (resistant) and Hokushin (susceptible) grown in a WYMV-infected nursery field were tested for the presence of WYMV in leaves by enzyme-linked immunosorbent assay (ELISA) and genotyped by using genome-wide molecular markers. Two major QTLs were detected: Qym1 located between Xgwm539 and Xgwm349 on chromosome 2DL and Qym2 located between Xbarc147 and Xwmc623 on chromosome 3BS. The resistance alleles for both QTLs originated from Madsen. The third QTL Qym3 located near Xwmc457 on chromosome 4D, where the resistant allele for this QTL originated from Hokushin. Although the Qym3 was rather minor, it was essential to complement Qym1 and Qym2 for complete avoidance of WYMV infection. Near-isogenic lines carrying the resistance QTLs were developed by repeated backcrosses using Madsen as the donor parent and Hokushin as the recurrent parent. The lines that were resistant to WYMV (as tested by ELISA) were homozygous for the Madsen alleles at both Qym1 and Qym2. Qym1 dominance was partial, whereas that of Qym2 was nearly complete. Qym1 was closely linked to Xwmc41; Qym2 was closely linked to Xwmc754. These markers will be useful in marker-assisted selection in wheat breeding for WYMV resistance; this study will facilitate cloning the WYMV resistance genes.
Assuntos
Resistência à Doença/genética , Vírus do Mosaico/patogenicidade , Doenças das Plantas/genética , Locos de Características Quantitativas , Triticum/genética , Alelos , Mapeamento Cromossômico , Cromossomos de Plantas , Cruzamentos Genéticos , DNA de Plantas/genética , Marcadores Genéticos , Genótipo , Endogamia , Modelos Lineares , Modelos Genéticos , Doenças das Plantas/virologia , Triticum/virologiaRESUMO
Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition we generated a single nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting especially activation of proinflammatory pathways. We further generated single nucleus multiomic data from four human AKI samples including validation by genome-wide identification of NF-kB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubule cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.
RESUMO
Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition, we generated a single-nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single-nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting, especially, activation of proinflammatory pathways. We further generated single-nucleus multiomic data from four human AKI samples including validation by genome-wide identification of nuclear factor κB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubular cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.
Assuntos
Injúria Renal Aguda , Epigênese Genética , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Camundongos , Humanos , Transcriptoma , NF-kappa B/metabolismo , NF-kappa B/genética , Modelos Animais de Doenças , Reprogramação Celular/genética , Proliferação de Células/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/metabolismoRESUMO
BACKGROUND: Mosaic loss of Y chromosome (LOY) is the most common chromosomal alteration in aging men. Here, we use single-cell RNA and ATAC sequencing to show that LOY is present in the kidney and increases with age and chronic kidney disease. RESULTS: The likelihood of a cell having LOY varies depending on its location in the nephron. Cortical epithelial cell types have a greater proportion of LOY than medullary or glomerular cell types, which may reflect their proliferative history. Proximal tubule cells are the most abundant cell type in the cortex and are susceptible to hypoxic injury. A subset of these cells acquires a pro-inflammatory transcription and chromatin accessibility profile associated with expression of HAVCR1, VCAM1, and PROM1. These injured epithelial cells have the greatest proportion of LOY and their presence predicts future kidney function decline. Moreover, proximal tubule cells with LOY are more likely to harbor additional large chromosomal gains and express pro-survival pathways. Spatial transcriptomics localizes injured proximal tubule cells to a pro-fibrotic microenvironment where they adopt a secretory phenotype and likely communicate with infiltrating immune cells. CONCLUSIONS: We hypothesize that LOY is an indicator of increased DNA damage and potential marker of cellular senescence that can be applied to single-cell datasets in other tissues.
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Cromossomos Humanos Y , Insuficiência Renal Crônica , Humanos , Masculino , Mosaicismo , Envelhecimento/genética , Fenótipo , Insuficiência Renal Crônica/genéticaRESUMO
Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.
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Injúria Renal Aguda , Proliferação de Células , Células Epiteliais , Proteína Forkhead Box M1 , Túbulos Renais Proximais , Animais , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box M1/genética , Camundongos , Proliferação de Células/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Masculino , Ciclinas/metabolismo , Ciclinas/genética , Camundongos Knockout , Modelos Animais de Doenças , Regulação da Expressão GênicaRESUMO
Emerging spatially resolved transcriptomics technologies allow for the measurement of gene expression in situ at cellular resolution. We apply direct RNA hybridization-based in situ sequencing (dRNA HybISS, Cartana part of 10xGenomics) to compare male and female healthy mouse kidneys and the male kidney injury and repair timecourse. A pre-selected panel of 200 genes is used to identify cell state dynamics patterns during injury and repair. We develop a new computational pipeline, CellScopes, for the rapid analysis, multi-omic integration and visualization of spatially resolved transcriptomic datasets. The resulting dataset allows us to resolve 13 kidney cell types within distinct kidney niches, dynamic alterations in cell state over the course of injury and repair and cell-cell interactions between leukocytes and kidney parenchyma. At late timepoints after injury, C3+ leukocytes are enriched near pro-inflammatory, failed-repair proximal tubule cells. Integration of snRNA-seq dataset from the same injury and repair samples also allows us to impute the spatial localization of genes not directly measured by dRNA HybISS.
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Rim , Transcriptoma , Camundongos , Animais , Masculino , Feminino , Rim/metabolismo , Transcriptoma/genética , Perfilação da Expressão Gênica/métodos , RNA/metabolismo , Túbulos Renais Proximais , Análise de Célula Única/métodosRESUMO
Renal proximal tubule epithelial cells have considerable intrinsic repair capacity following injury. However, a fraction of injured proximal tubule cells fails to undergo normal repair and assumes a proinflammatory and profibrotic phenotype that may promote fibrosis and chronic kidney disease. The healthy to failed repair change is marked by cell state-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq sequencing offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We develop a regularized regression approach to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generate a single nucleus multiomic dataset from seven adult human kidney samples and apply our method to study drivers of a failed injury response associated with kidney disease. We demonstrate that our approach is a highly effective tool for predicting key cis- and trans-regulatory elements underpinning the healthy to failed repair transition and use it to identify NFAT5 as a driver of the maladaptive proximal tubule state.
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Multiômica , Insuficiência Renal Crônica , Adulto , Humanos , Rim , Túbulos Renais Proximais , Células EpiteliaisRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.
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The Ppd-A1 genotype of 240 Japanese wheat cultivars and 40 foreign cultivars was determined using a PCR-based method. Among Japanese cultivars, only 12 cultivars, all of which were Hokkaido winter wheat, carried the Ppd-A1a allele, while this allele was not found in Hokkaido spring wheat cultivars or Tohoku-Kyushu cultivars. Cultivars with a photoperiod-insensitive allele headed 6.9-9.8 days earlier in Kanto and 2.5 days earlier in Hokkaido than photoperiod-sensitive cultivars. The lower effect of photoperiod-insensitive alleles observed in Hokkaido could be due to the longer day-length at the spike formation stage compared with that in Kanto. Pedigree analysis showed that 'Purple Straw' and 'Tohoku 118' were donors of Ppd-A1a and Ppd-D1a in Hokkaido wheat cultivars, respectively. Wheat cultivars recently developed in Hokkaido carry photoperiod-insensitive alleles at a high frequency. For efficient utilization of Ppd-1 alleles in the Hokkaido wheat-breeding program, the effect of Ppd-1 on growth pattern and grain yield should be investigated. Ppd-A1a may be useful as a unique gene source for fine tuning the heading time in the Tohoku-Kyushu region since the effect of Ppd-A1a on photoperiod insensitivity appears to differ from the effect of Ppd-B1a and Ppd-D1a.
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Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.
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Cistos , Rim Policístico Autossômico Dominante , Humanos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Análise de Célula Única , Rim/metabolismo , Túbulos Renais/metabolismo , Células Epiteliais/metabolismo , Cistos/metabolismo , Receptores Acoplados a Proteínas G/metabolismoAssuntos
Neuropatias Amiloides Familiares/genética , Achados Incidentais , Mutação , Pré-Albumina/genética , Proteinúria/genética , Urinálise , Idoso , Neuropatias Amiloides Familiares/complicações , Neuropatias Amiloides Familiares/diagnóstico , Neuropatias Amiloides Familiares/urina , Biópsia , Cromatografia Líquida , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Rim/química , Rim/patologia , Fenótipo , Pré-Albumina/análise , Valor Preditivo dos Testes , Proteinúria/diagnóstico , Proteinúria/urina , Espectrometria de Massas em TandemRESUMO
Maximizing the potential of human kidney organoids for drug testing and regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a mature collecting system, and off-target cell types. By independently generating two kidney progenitor cell populations-metanephric mesenchyme and ureteric bud (UB)-like cells-we could generate kidney organoids with a collecting system. We also identify the hormones aldosterone and arginine vasopressin (AVP) as critical to promote differentiation of collecting duct cell types including both principal cells (PCs) and intercalated cells (ICs). The resulting PCs express aquaporin-2 (AQP2) protein, which undergoes translocation to the apical membrane after vasopressin or forskolin stimulation. By single-cell RNA sequencing (scRNA-seq), we demonstrate improved proximal tubule maturation and reduced off-target cell populations. We also show appropriate downregulation of progenitor cell types, improved modeling of tubular injury, the presence of urothelium (Uro), and the ability of Notch pathway modulation to regulate PC:IC ratios during organoid development.
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Túbulos Renais/metabolismo , Rim/patologia , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , HumanosRESUMO
Nephrons, the functional units of the kidney, are derived from nephron progenitor cells (NPCs). Here, we describe methods to reconstruct nephron tissue via induction of NPCs from mouse and human pluripotent stem cells, which mimic multistep developmental signals in vivo. Induced NPCs differentiate into three-dimensional nephron structures, including glomerular podocytes and nephric tubules, which are useful for studying early stages of kidney specification and morphogenetic processes in the context of normal development or disease.