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1.
Artículo en Inglés | MEDLINE | ID: mdl-39250817

RESUMEN

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, co-immunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology.

2.
J Clin Invest ; 134(18)2024 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-39286973

RESUMEN

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury-responsive fibroblast subset defined by aberrant high JAK/STAT signaling.


Asunto(s)
Matriz Extracelular , Fibrosis , Ratones Noqueados , Animales , Ratones , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Fibroblastos/metabolismo , Fibroblastos/patología , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Transducción de Señal , Miofibroblastos/metabolismo , Miofibroblastos/patología
3.
Am J Pathol ; 2024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-39097166

RESUMEN

The recent arrival of high-resolution spatial transcriptomics (ST) technologies is generating a veritable revolution in life sciences, enabling biomolecules to be measured in their native spatial context. By integrating morphology and molecular biology, ST technologies offer the potential of improving the understanding of tissue biology and disease and may also provide meaningful clinical insights. In this review, we describe the main ST technologies currently available and the computational analysis for data interpretation and visualization, and illustrate their scientific and potential medical interest in the context of kidney disease. Finally, we discuss the perspectives and challenges of these booming new technologies.

4.
Sci Adv ; 10(32): eado2849, 2024 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-39110788

RESUMEN

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.


Asunto(s)
Lesión Renal Aguda , Epigénesis Genética , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Ratones , Humanos , Transcriptoma , FN-kappa B/metabolismo , FN-kappa B/genética , Modelos Animales de Enfermedad , Reprogramación Celular/genética , Proliferación Celular/genética , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/metabolismo
5.
Am J Pathol ; 2024 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-39097165

RESUMEN

The application of spatial transcriptomics (ST) technologies is booming and has already yielded important insights across many different tissues and disease models. In nephrology, ST technologies have helped to decipher the cellular and molecular mechanisms at work in kidney diseases and have allowed the recent creation of spatially anchored human kidney atlases in healthy and diseased kidney tissues. During ST data analysis, the obtained computationally annotated clusters are often superimposed on a histologic image without their initial identification being based on the morphologic and spatial analyses of the tissues and lesions. In this study, we conduct a histopathologic-based analysis of ST data on a human kidney sample corresponding as closely as possible to the reality of the interpretation of a kidney biopsy sample in a health care or research context. This study shows the feasibility of a morphology-based approach to interpreting ST data, helping to improve our understanding of the lesion phenomena at work in chronic kidney disease at both the cellular and the molecular level. Finally, we show that our newly identified pathology-based clusters can be accurately projected onto other slides from nephrectomy or needle biopsy samples. They thus serve as a reference for analyzing other kidney tissues, paving the way for the future of molecular microscopy and precision pathology.

6.
Kidney Int ; 2024 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-39009187
8.
Sci Data ; 11(1): 797, 2024 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-39025878

RESUMEN

The three major anatomic regions of the human kidney include the cortex, medulla and papilla, with different functions and vulnerabilities to kidney diseases. Epigenetic mechanisms underlying these anatomic structures are incompletely understood. Here, we performed chromatin conformation capture with Hi-C and histone modification H3K4me3/H3K27me3 Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing on the kidney cortex, medulla and papilla dissected from one individual donor. Nuclear suspensions were generated from each region and split subjected to paired Hi-C and CUT&RUN sequencing. We evaluated the quality of next-generation sequencing data, Hi-C chromatin contact matrices and CUT&RUN peak calling. H3K4me3 and H3K27me3 histone modifications represent active and repressive gene transcription, respectively, and differences in chromatin conformation between kidney regions can be analyzed with this dataset. All raw and processed data files are publicly available, allowing researchers to survey the epigenetic landscape across regional human kidney anatomy.


Asunto(s)
Cromatina , Histonas , Riñón , Humanos , Cromatina/metabolismo , Histonas/metabolismo , Riñón/metabolismo , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Código de Histonas
9.
STAR Protoc ; 5(3): 103049, 2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38900631

RESUMEN

Simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) profiles transcriptomics and chromatin accessibility in the same cells at high throughput. Here, we present a protocol for multimodal profiling of human kidneys with SHARE-seq. We describe steps for processing fixed nuclei for SHARE-seq split-pool barcoding and library preparation. We also detail how to determine the optimal working concentration of Tn5 transposase for transposition and tagmentation. This protocol allows researchers to generate large-scale single-cell multiomics data at low reagent cost. For complete details on the use and execution of this protocol, please refer to Li et al.1.

11.
bioRxiv ; 2024 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-38854144

RESUMEN

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.

12.
JCI Insight ; 9(15)2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38916959

RESUMEN

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.


Asunto(s)
Lesión Renal Aguda , Proliferación Celular , Células Epiteliales , Proteína Forkhead Box M1 , Túbulos Renales Proximales , Animales , Masculino , Ratones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/genética , Proliferación Celular/genética , Ciclinas/metabolismo , Ciclinas/genética , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box M1/genética , Regulación de la Expresión Génica , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/citología , Ratones Noqueados
13.
Kidney Int ; 2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38901605

RESUMEN

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

15.
Data Brief ; 54: 110431, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38708307

RESUMEN

Cortex, medulla and papilla are three major human kidney anatomic structures and they harbour unique metabolic functions, but the underlying metabolomic profiles are largely unknown at spatial resolution. Here, we generated a spatially resolved metabolomics dataset on human kidney cortex, medulla and papilla tissues dissected from the same donor. Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry (MALDI-IMS) was used to detect metabolite species over mass-to-charge ratios of 50 -1500 for each section at a resolution of 10 × 10 µm2 pixel size. We present raw data matrix of each sample, feature annotations, raw AnnData merged from three samples and processed AnnData files after quality control, dimensional reduction and data integration, which contains a total of 170,459 spatially resolved metabolomes with 562 features detected. This dataset can be either visualized through an interactive browser or further analyzed to study metabolomic heterogeneity across regional human kidney anatomy.

16.
Am J Physiol Renal Physiol ; 326(5): F827-F838, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38482555

RESUMEN

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.


Asunto(s)
Lesión Renal Aguda , Apoptosis , Células Epiteliales , Túbulos Renales Proximales , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Animales , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 2 Asociado a Receptor de TNF/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/genética , Transducción de Señal , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Línea Celular , Inflamación/metabolismo , Inflamación/patología , Masculino
18.
JCI Insight ; 9(6)2024 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-38516886

RESUMEN

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.


Asunto(s)
Carnitina O-Palmitoiltransferasa , Riñón , Animales , Ratones , Envejecimiento/genética , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Riñón/metabolismo , Riñón/patología , Túbulos Renales/metabolismo
19.
Cell Metab ; 36(5): 1105-1125.e10, 2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38513647

RESUMEN

A large-scale multimodal atlas that includes major kidney regions is lacking. Here, we employed simultaneous high-throughput single-cell ATAC/RNA sequencing (SHARE-seq) and spatially resolved metabolomics to profile 54 human samples from distinct kidney anatomical regions. We generated transcriptomes of 446,267 cells and chromatin accessibility profiles of 401,875 cells and developed a package to analyze 408,218 spatially resolved metabolomes. We find that the same cell type, including thin limb, thick ascending limb loop of Henle and principal cells, display distinct transcriptomic, chromatin accessibility, and metabolomic signatures, depending on anatomic location. Surveying metabolism-associated gene profiles revealed non-overlapping metabolic signatures between nephron segments and dysregulated lipid metabolism in diseased proximal tubule (PT) cells. Integrating multimodal omics with clinical data identified PLEKHA1 as a disease marker, and its in vitro knockdown increased gene expression in PT differentiation, suggesting possible pathogenic roles. This study highlights previously underrepresented cellular heterogeneity underlying the human kidney anatomy.


Asunto(s)
Epigenómica , Riñón , Metabolómica , Transcriptoma , Humanos , Riñón/metabolismo , Masculino , Perfilación de la Expresión Génica , Femenino
20.
bioRxiv ; 2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38328130

RESUMEN

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.

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