RESUMEN
In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo-producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP-derived (REPD) cell lines. Under nonpermissive conditions, REPD cells ceased from proliferation and acquired a stem cell-like state, with strongly enhanced hypoxia-inducible factor 2 (HIF-2α), stem cell antigen 1 (SCA-1), and CD133 expression, but also enhanced alpha-smooth muscle actin (αSMA) expression, indicating myofibroblastic signaling. These cells maintained the "on-off" nature of Epo expression observed in REP cells in vivo, whereas other HIF target genes showed a more permanent regulation. Like REP cells in vivo, REPD cells cultured in vitro generated long tunneling nanotubes (TNTs) that aligned with endothelial vascular structures, were densely packed with mitochondria and became more numerous under hypoxic conditions. Although inhibition of mitochondrial oxygen consumption blunted HIF signaling, removal of the TNTs did not affect or even enhance the expression of HIF target genes. Apart from pericytes, REPD cells readily differentiated into neuroglia but not adipogenic, chondrogenic, or osteogenic lineages, consistent with a neuronal origin of at least a subpopulation of REP cells. In summary, these results suggest an unprecedented combination of differentiation features of this unique cell type.
Asunto(s)
Eritropoyetina , Pericitos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Eritropoyesis , Eritropoyetina/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Riñón/metabolismo , Mamíferos/metabolismo , Ratones , Ratones Transgénicos , Pericitos/metabolismoRESUMEN
Renal erythropoietin (Epo)-producing (REP) cells represent a rare and incompletely understood cell type. REP cells are fibroblast-like cells located in close proximity to blood vessels and tubules of the corticomedullary border region. Epo mRNA in REP cells is produced in a pronounced "on-off" mode, showing transient transcriptional bursts upon exposure to hypoxia. In contrast to "ordinary" fibroblasts, REP cells do not proliferate ex vivo, cease to produce Epo, and lose their identity following immortalization and prolonged in vitro culture, consistent with the loss of Epo production following REP cell proliferation during tissue remodelling in chronic kidney disease. Because Epo protein is usually not detectable in kidney tissue, and Epo mRNA is only transiently induced under hypoxic conditions, transgenic mouse models have been developed to permanently label REP cell precursors, active Epo producers, and inactive descendants. Future single-cell analyses of the renal stromal compartment will identify novel characteristic markers of tagged REP cells, which will provide novel insights into the regulation of Epo expression in this unique cell type.
Asunto(s)
Eritropoyetina , Insuficiencia Renal Crónica , Animales , Eritropoyetina/metabolismo , Hipoxia/metabolismo , Riñón/metabolismo , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/metabolismoRESUMEN
Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cisteína Endopeptidasas/genética , Eliminación de Gen , Resistencia a la Insulina/genética , Insulina/administración & dosificación , Oxigenasas de Función Mixta/metabolismo , Adenilato Quinasa/metabolismo , Animales , Glucemia , Peso Corporal , Tamaño de la Célula , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Metabolismo Energético , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Insulina/efectos adversos , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.
Asunto(s)
Eritropoyetina/metabolismo , Telocitos/patología , Factores de Transcripción/metabolismo , Anemia/etiología , Anemia/patología , Animales , Hipoxia de la Célula , Línea Celular , Eritropoyetina/genética , Retroalimentación Fisiológica , Riñón/citología , Riñón/patología , Ratones , Ratones Transgénicos , Cultivo Primario de Células , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/patología , Telocitos/metabolismoRESUMEN
Erythropoietin (Epo) is the master regulator of erythropoiesis and oxygen homeostasis. Despite its physiological importance, the molecular and genomic contexts of the cells responsible for renal Epo production remain unclear, limiting more-effective therapies for anemia. Here, we performed single-cell RNA and transposase-accessible chromatin (ATAC) sequencing of an Epo reporter mouse to molecularly identify Epo-producing cells under hypoxic conditions. Our data indicate that a distinct population of kidney stroma, which we term Norn cells, is the major source of endocrine Epo production in mice. We use these datasets to identify the markers, signaling pathways and transcriptional circuits characteristic of Norn cells. Using single-cell RNA sequencing and RNA in situ hybridization in human kidney tissues, we further provide evidence that this cell population is conserved in humans. These preliminary findings open new avenues to functionally dissect EPO gene regulation in health and disease and may serve as groundwork to improve erythropoiesis-stimulating therapies.
Asunto(s)
Anemia , Eritropoyetina , Animales , Humanos , Ratones , Anemia/genética , Eritropoyesis/genética , Eritropoyetina/genética , Riñón/metabolismo , ARN/metabolismoRESUMEN
One of the widely used applications of the popular Cre-loxP method for targeted recombination is the permanent activation of marker genes, such as reporter genes or antibiotic resistance genes, by excision of a preceding transcriptional stop signal. The STOP cassette consists of three identical SV40-derived poly(A) signal repeats and is flanked by two loxP sites. We found that in addition to complete loxP-mediated recombination, limiting levels of the Cre recombinase also cause incomplete recombination of the STOP cassette. Partial recombination leads to the loss of only one or two of the three identical poly(A) repeats with recombination breakpoints always precisely matching the end/start of each poly(A) signal repeat without any relevant similarity to the canonical or known cryptic loxP sequences, suggesting that this type of Cre-mediated recombination is loxP-independent. Incomplete deletion of the STOP cassette results in partial read-through transcription, explaining at least some of the variability often observed in marker gene expression from an otherwise identical locus.
Asunto(s)
Integrasas/genética , Integrasas/metabolismo , Recombinación Genética , Animales , Células CHO , Cricetulus , Femenino , Expresión Génica , Genes Reporteros/genética , Marcadores Genéticos , Riñón , Masculino , Ratones , TranscriptomaRESUMEN
Netherton syndrome is a monogenic autosomal recessive disorder primarily characterized by the detachment of the uppermost layer of the epidermis, the stratum corneum It results from mutations in the SPINK5 gene, which codes for a kallikrein inhibitor. Uncontrolled kallikrein activity leads to premature desquamation, resulting in a severe epidermal barrier defect and subsequent life-threatening systemic infections and chronic cutaneous inflammation. Here, we show that genetic activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2/Nrf2) in keratinocytes of Spink5 knockout mice, a model for Netherton syndrome, significantly alleviates their cutaneous phenotype. Nrf2 activation promoted attachment of the stratum corneum and concomitant epidermal barrier function, and reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α and thymic stromal lymphopoietin. Mechanistically, we show that Nrf2 activation induces overexpression of secretory leukocyte protease inhibitor (Slpi), a known inhibitor of kallikrein 7 and elastase 2, in mouse and human keratinocytes in vivo and in vitro, respectively. In the Spink5-deficient epidermis, the upregulation of Slpi is likely to promote stabilization of corneodesmosomes, thereby preventing premature desquamation. Our results suggest pharmacological NRF2 activation as a promising treatment modality for Netherton syndrome patients.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Factor 2 Relacionado con NF-E2/genética , Síndrome de Netherton/genética , Síndrome de Netherton/patología , Piel/patología , Animales , Adhesión Celular , Diferenciación Celular , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Epidermis/patología , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Integrasas/metabolismo , Queratinocitos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor de Serinpeptidasas Tipo Kazal-5/deficiencia , Inhibidor de Serinpeptidasas Tipo Kazal-5/genéticaRESUMEN
We investigated molecular and cellular parameters which set metabolic and mechanical functioning of knee extensor muscles in the operated and contralateral control leg of 9 patients with a chronically insufficient anterior cruciate ligament (ACL; 26.6 ± 8.3 years, 8 males, 1 female) after open reconstructive surgery (week 0), after ambulant physiotherapy under cast immobilization (week 9), succeeding rehabilitation training (up to week 26), and subsequent voluntary physical activity (week 260). Clinical indices of knee function in the operated leg were improved at 52 weeks and remained at a comparable level at week 260. CSA of the quadriceps (-18%), MCSA of muscle fibers (-24%), and capillary-to-fiber ratio (-24%) in m. vastus lateralis from the ACL insufficient leg were lower at week 0 than reference values in the contralateral leg at week 260. Slow type fiber percentage (-35%) and mitochondrial volume density (-39%) were reduced in m. vastus lateralis from the operated leg at weeks 9 and 26. Composition alterations in the operated leg exceeded those in the contralateral leg and, with the exception of the volume density of subsarcolemmal mitochondria, returned to the reference levels at week 260. Leg-specific deterioration of metabolic characteristics in the vasti from the operated leg was reflected by the down-regulation of mitochondrial respiration complex I-III markers (-41-57%) at week 9. After rehabilitation training at week 26, the specific Y397 phosphorylation of focal adhesion kinase (FAK), which is a proxy for mechano-regulation, was elevated by 71% in the operated leg but not in the contralateral leg, which had performed strengthening type exercise during ambulant physiotherapy. Total FAK protein and Y397 phosphorylation levels were lowered in both legs at week 26 resulting in positive correlations with mitochondrial volume densities and mitochondrial protein levels. The findings emphasize that a loss of mechanical and metabolic characteristics in knee extensor muscle remains detectable years after untreated ACL rupture, which may be aggravated in the post-operative phase by the deterioration of slow-oxidative characteristics after reconstruction due to insufficient load-bearing muscle activity. The reestablishment of muscle composition subsequent to years of voluntary physical activity reinforces that slow-to-fast fiber transformation is reversible in humans.