RESUMO
BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disease caused by mutations in the galactosidase α gene. Deficient activity of α-galactosidase A leads to glycosphingolipid accumulations in multiple organs. Circular RNAs represent strong regulators of gene expression. Their circular structure ensures high stability in blood. We hypothesised that blood-based circular RNA profiles improve phenotypic assignment and therapeutic monitoring of Fabry disease. METHODS: A genome-wide circular RNA expression analysis was performed in blood of genetically diagnosed patients with Fabry disease (n=58), age-matched and sex-matched healthy volunteers (n=14) and disease control patients with acute kidney injury (n=109). Most highly dysregulated circular RNAs were validated by quantitative real-time PCR. Circular RNA biomarker sensitivity, specificity, predictive values and area under the curve (AUC) were determined. Linear regression analyses were conducted for validated circular RNA biomarkers and clinical patient characteristics. RESULTS: A distinct circular RNA transcriptome signature identified patients with Fabry disease. Level of circular RNAs hsa_circ_0006853 (AUC=0.73), hsa_circ_0083766 (AUC=0.8) and hsa_circ_0002397 (AUC=0.8) distinguished patients with Fabry disease from both healthy controls and patients with acute kidney injury. Hsa_circ_0002397 was, furthermore, female-specifically expressed. Circular RNA level were significantly related to galactosidase α gene mutations, early symptoms, phenotypes, disease severities, specific therapies and long-term complications of Fabry disease. CONCLUSION: The discovery of circular RNA-based and Fabry disease-specific biomarkers may advance future diagnosis of Fabry disease and help to distinguish related phenotypes.
Assuntos
Injúria Renal Aguda , Doença de Fabry , Biomarcadores/metabolismo , Biomarcadores Tumorais , Doença de Fabry/diagnóstico , Doença de Fabry/genética , Feminino , Galactosidases/genética , Humanos , Masculino , Fenótipo , RNA/genética , RNA/metabolismo , RNA Circular/genéticaRESUMO
Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG "tomato" or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.
Assuntos
Injúria Renal Aguda/patologia , Ciclo Celular , Separação Celular/métodos , Células Epiteliais/patologia , Citometria de Fluxo , Túbulos Renais Proximais/patologia , Fixação de Tecidos/métodos , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Animais , Ácidos Aristolóquicos , Biomarcadores/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Genes Reporter , Túbulos Renais Proximais/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Transgênicos , PoliploidiaRESUMO
The TGF-ß and Wnt/ß-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF-ß has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF-ß signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF-ß type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF-ß receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF-ß receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/ß-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF-ß receptor impaired ß-catenin activity in vitro and in vivo Genetically restoring ß-catenin activity in proximal tubules lacking the TGF-ß receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF-ß receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of ß-catenin activity or an indirect effect of ß-catenin interacting with other growth factors. In conclusion, blocking TGF-ß and ß-catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Assuntos
Falência Renal Crônica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/metabolismo , Animais , Ácidos Aristolóquicos/química , Núcleo Celular/metabolismo , Colágeno/química , Cruzamentos Genéticos , Epitélio/metabolismo , Feminino , Deleção de Genes , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidoresRESUMO
Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) play key roles in regulating the response to renal injury but are thought to mediate divergent effects on cell behavior. However, how TGF-ß signaling alters the response to HGF in epithelia, the key site of HGF signaling in the injured kidney, is not well studied. Contrary to our expectation, we showed that deletion of the TGF-ß type II receptor in conditionally immortalized proximal tubule (PT) cells impaired HGF-dependent signaling. This reduced signaling was due to decreased transcription of c-Met, the HGF receptor, and the TGF-ß-dependent c-Met transcription and increased response to HGF in PT cells were mediated by the Notch pathway. The interactions of TGF-ß, HGF, and Notch pathways had biologically significant effects on branching morphogenesis, cell morphology, migration, and proliferation. In conclusion, epithelial TGF-ß signaling promotes HGF signaling in a Notch-dependent pathway. These findings suggest that TGF-ß modulates PT responses not only by direct effects, but also by affecting other growth factor signaling pathways.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Células Cultivadas , Camundongos , Proteínas Serina-Treonina Quinases/deficiência , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
Transforming growth factor-ß (TGF-ß) strongly promotes renal tubulointerstitial fibrosis, but the cellular target that mediates its profibrotic actions has not been clearly identified. While in vitro data suggest that TGF-ß-induced matrix production is mediated by renal fibroblasts, the role of these cells in TGF-ß-dependent tubulointerstitial fibrosis following renal injury is not well defined. To address this, we deleted the TGF-ß type II receptor in matrix-producing interstitial cells using two different inducible Cre models: COL1A2-Cre with a mesenchymal enhancer element and tenascin-Cre that targets medullary interstitial cells, and either the mouse unilateral ureteral obstruction or the aristolochic acid renal injury model. Renal interstitial cells lacking the TGF-ß receptor had significantly impaired collagen I production, but, unexpectedly, overall tissue fibrosis was unchanged in the conditional knockouts after renal injury. Thus, abrogating TGF-ß signaling in matrix-producing interstitial cells is not sufficient to reduce fibrosis after renal injury.
Assuntos
Matriz Extracelular/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Animais , Ácidos Aristolóquicos , Células Cultivadas , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/genética , Fibrose , Rim/patologia , Nefropatias/etiologia , Nefropatias/genética , Nefropatias/patologia , Nefropatias/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Tempo , Obstrução Ureteral/complicaçõesRESUMO
Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-ß1 (TGF-ß1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1 cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-ß1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.
Assuntos
Proteínas de Fase Aguda/fisiologia , Albuminas/metabolismo , Albuminúria/metabolismo , Albuminúria/patologia , Túbulos Renais Coletores/patologia , Lipocalinas/fisiologia , Proteínas Oncogênicas/fisiologia , Albuminúria/complicações , Animais , Linhagem Celular , Endocitose/fisiologia , Túbulos Renais Coletores/metabolismo , Lipocalina-2 , Masculino , Camundongos , NF-kappa B/metabolismo , Nefrite/etiologia , Nefrite/metabolismo , Nefroesclerose/etiologia , Nefroesclerose/metabolismo , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta1/metabolismoRESUMO
NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD(cl1)) cell line increased TGF-ß1-induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.
Assuntos
Nefropatias/enzimologia , Rim/patologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose , Atrofia , Capilares/patologia , Fibrose , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidase 4 , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Obstrução Ureteral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nephrotoxicity is a major cause of kidney disease and failure in drug development, but understanding of cellular mechanisms is limited, highlighting the need for better experimental models and methodological approaches. Most nephrotoxins damage the proximal tubule (PT), causing functional impairment of solute reabsorption and systemic metabolic complications. The antiviral drug tenofovir disoproxil fumarate (TDF) is an archetypal nephrotoxin, inducing mitochondrial abnormalities and urinary solute wasting, for reasons that were previously unclear. Here, we developed an automated, high-throughput imaging pipeline to screen the effects of TDF on solute transport and mitochondrial morphology in human-derived RPTEC/TERT1 cells, and leveraged this to generate realistic models of functional toxicity. By applying multiparametric metabolic profiling-including oxygen consumption measurements, metabolomics, and transcriptomics-we elucidated a highly robust molecular fingerprint of TDF exposure. Crucially, we identified that the active metabolite inhibits complex V (ATP synthase), and that TDF treatment causes rapid, dose-dependent loss of complex V activity and expression. Moreover, we found evidence of complex V suppression in kidney biopsies from humans with TDF toxicity. Thus, we demonstrate an effective and convenient experimental approach to screen for disease relevant functional defects in kidney cells in vitro, and reveal a new paradigm for understanding the pathogenesis of a substantial cause of nephrotoxicity.
Assuntos
Antivirais , Insuficiência Renal , Humanos , Tenofovir/efeitos adversos , Antivirais/metabolismo , Rim , Mitocôndrias , Insuficiência Renal/tratamento farmacológico , MetabolômicaRESUMO
The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.
Assuntos
Proteína Forkhead Box O3/metabolismo , Túbulos Renais Proximais/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Proteína Forkhead Box O3/genética , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , beta Catenina/genéticaRESUMO
NADPH oxidase 4 (NOX4) is highly expressed in kidney proximal tubular cells. NOX4 constitutively produces hydrogen peroxide, which may regulate important pro-survival pathways. Renal ischemia reperfusion injury (IRI) is a classical model mimicking human ischemic acute tubular necrosis. We hypothesized that NOX4 plays a protective role in kidney IRI. In wild type (WT) animals subjected to IRI, NOX4 protein expression increased after 24 hours. NOX4 KO (knock-out) and WT littermates mice were subjected to IRI. NOX4 KO mice displayed decreased renal function and more severe tubular apoptosis, decreased Bcl-2 expression and higher histologic damage scores compared to WT. Activation of NRF2 was decreased in NOX4 KO mice in response to IRI. This was related to decreased KEAP1 oxidation leading to decreased NRF2 stabilization. This resulted in decreased glutathione levels. In vitro silencing of NOX4 in cells showed an enhanced propensity to apoptosis, with reduced expression of NRF2, glutathione content and Bcl-2 expression, similar to cells derived from NOX4 KO mice. Overexpression of a constitutively active form of NRF2 (caNRF2) in NOX4 depleted cells rescued most of this phenotype in cultured cells, implying that NRF2 regulation by ROS issued from NOX4 may play an important role in its anti-apoptotic property.