RESUMEN
The main laminin-binding integrins α3ß1, α6ß1 and α6ß4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.
Asunto(s)
Integrinas , Túbulos Renales Colectores , Animales , Células Epiteliales , Inflamación/genética , Integrina alfa3beta1 , Integrinas/genética , Laminina/genética , Ratones , FN-kappa B/genéticaRESUMEN
A protective role for sirtuin 6 (Sirt6) in the context of chronic renal injury is reported by Cai et al. in this issue of Kidney International. The mechanism is thought to be mediated by Sirt6's deacetylase activity, specifically on ß-catenin target genes. This commentary discusses these results and the interaction between Sirt6 and ß-catenin within the broader context of ß-catenin signaling and injury.
Asunto(s)
Enfermedades Renales , beta Catenina , Fibrosis , Humanos , Riñón , SirtuinasRESUMEN
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.
Asunto(s)
Lesión Renal Aguda/patología , Ciclo Celular , Separación Celular/métodos , Células Epiteliales/patología , Citometría de Flujo , Túbulos Renales Proximales/patología , Fijación del Tejido/métodos , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Animales , Ácidos Aristolóquicos , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Genes Reporteros , Túbulos Renales Proximales/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones Transgénicos , PoliploidíaRESUMEN
Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Fibrosis/patología , Enfermedades Renales Poliquísticas/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Linaje de la Célula , Femenino , Fibrosis/genética , Túbulos Renales Proximales/patología , Masculino , Ratones , Miofibroblastos/fisiología , Enfermedades Renales Poliquísticas/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Epidermal growth factor receptor (EGFR) has been implicated in the pathogenesis of diabetic nephropathy and renal fibrosis; however, the causative role of sustained EGFR activation is unclear. Here, we generated a novel kidney fibrotic mouse model of persistent EGFR activation by selectively expressing the EGFR ligand, human heparin-binding EGF-like growth factor (hHB-EGF), in renal proximal tubule epithelium. hHB-EGF expression increased tyrosine kinase phosphorylation of EGFR and the subsequent activation of downstream signaling pathways, including ERK and AKT, as well as the profibrotic TGF-ß1/SMAD pathway. Epithelial-specific activation of EGFR was sufficient to promote spontaneous and progressive renal tubulointerstitial fibrosis, as characterized by increased collagen deposition, immune cell infiltration, and α-smooth muscle actin (α-SMA)-positive myofibroblasts. Tubule-specific EGFR activation promoted epithelial dedifferentiation and cell-cycle arrest. Furthermore, EGFR activation in epithelial cells promoted the proliferation of α-SMA+ myofibroblasts in a paracrine manner. Genetic or pharmacologic inhibition of EGFR tyrosine kinase activity or downstream MEK activity attenuated the fibrotic phenotype. This study provides definitive evidence that sustained activation of EGFR in proximal epithelia is sufficient to cause spontaneous, progressive renal tubulointerstitial fibrosis, evident by epithelial dedifferentiation, increased myofibroblasts, immune cell infiltration, and increased matrix deposition.-Overstreet, J. M., Wang, Y., Wang, X., Niu, A., Gewin, L. S., Yao, B., Harris, R. C., Zhang, M.-Z. Selective activation of epidermal growth factor receptor in renal proximal tubule induces tubulointerstitial fibrosis.
Asunto(s)
Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Túbulos Renales Proximales/metabolismo , Animales , Puntos de Control del Ciclo Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibrosis/metabolismo , Humanos , Riñón/metabolismo , Túbulos Renales Proximales/citología , Ratones Transgénicos , Miofibroblastos/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Fallo Renal Crónico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , beta Catenina/metabolismo , Animales , Ácidos Aristolóquicos/química , Núcleo Celular/metabolismo , Colágeno/química , Cruzamientos Genéticos , Epitelio/metabolismo , Femenino , Eliminación de Gen , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inhibidoresAsunto(s)
Hipoxia , Insuficiencia Renal Crónica , Eritrocitos , Humanos , Lisofosfolípidos , Esfingosina/análogos & derivadosRESUMEN
Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.
Asunto(s)
Apoptosis/fisiología , Células Epiteliales/metabolismo , Sistema de Señalización de MAP Quinasas , Transducción de Señal , Análisis de la Célula Individual , Factor de Necrosis Tumoral alfa/fisiología , Activación Enzimática , HumanosRESUMEN
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/metabolismoRESUMEN
Acute and chronic kidney injuries induce increased cell cycle progression in renal tubules. While increased cell cycle progression promotes repair after acute injury, the role of ongoing tubular cell cycle progression in chronic kidney disease is unknown. Two weeks after initiation of chronic kidney disease, we blocked cell cycle progression at G1/S phase by using an FDA-approved, selective inhibitor of CDK4/6. Blocking CDK4/6 improved renal function and reduced tubular injury and fibrosis in 2 murine models of chronic kidney disease. However, selective deletion of cyclin D1, which complexes with CDK4/6 to promote cell cycle progression, paradoxically increased tubular injury. Expression quantitative trait loci (eQTLs) for CCND1 (cyclin D1) and the CDK4/6 inhibitor CDKN2B were associated with eGFR in genome-wide association studies. Consistent with the preclinical studies, reduced expression of CDKN2B correlated with lower eGFR values, and higher levels of CCND1 correlated with higher eGFR values. CDK4/6 inhibition promoted tubular cell survival, in part, through a STAT3/IL-1ß pathway and was dependent upon on its effects on the cell cycle. Our data challenge the paradigm that tubular cell cycle progression is beneficial in the context of chronic kidney injury. Unlike the reparative role of cell cycle progression following acute kidney injury, these data suggest that blocking cell cycle progression by inhibiting CDK4/6, but not cyclin D1, protects against chronic kidney injury.
Asunto(s)
Ciclina D1 , Insuficiencia Renal Crónica , Animales , Ciclo Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Estudio de Asociación del Genoma Completo , Ratones , Insuficiencia Renal Crónica/tratamiento farmacológicoRESUMEN
The kidney is a highly metabolically active organ that relies on specialized epithelial cells comprising the renal tubules to reabsorb most of the filtered water and solutes. Most of this reabsorption is mediated by the proximal tubules, and high amounts of energy are needed to facilitate solute movement. Thus, proximal tubules use fatty acid oxidation, which generates more adenosine triphosphate (ATP) than glucose metabolism, as its preferred metabolic pathway. After kidney injury, metabolism is altered, leading to decreased fatty acid oxidation and increased lactic acid generation. This review discusses how metabolism differs between the proximal and more distal tubular segments of the healthy nephron. In addition, metabolic changes in acute kidney injury and chronic kidney disease are discussed, as well as how these changes in metabolism may impact tubule repair and chronic kidney disease progression.
Asunto(s)
Lesión Renal Aguda/metabolismo , Azúcares de la Dieta/metabolismo , Ácidos Grasos/metabolismo , Túbulos Renales/metabolismo , Insuficiencia Renal Crónica/metabolismo , Progresión de la Enfermedad , Humanos , Túbulos Renales Proximales/metabolismo , Ácido Láctico/biosíntesis , Oxidación-ReducciónRESUMEN
A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2-Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.
Asunto(s)
Actomiosina/metabolismo , Túbulos Renales Colectores/metabolismo , Neuropéptidos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Polaridad Celular/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Miosina Tipo II/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Acute kidney injury (AKI) portends a poor clinical prognosis and increases the risk for the development of chronic kidney disease (CKD). Currently, there are no therapies to treat AKI or prevent its progression to CKD. Wnt/ß-catenin is a critical regulator of kidney development that is up-regulated after injury. Most of the literature support a beneficial role for Wnt/ß-catenin in AKI, but suggest that this pathway promotes the progression of tubulointerstitial fibrosis, the hallmark of CKD progression. We review the role of Wnt/ß-catenin in renal injury with a focus on its potential as a therapeutic target in AKI and in AKI to CKD transition.
Asunto(s)
Lesión Renal Aguda/metabolismo , Túbulos Renales/metabolismo , Insuficiencia Renal Crónica/metabolismo , Vía de Señalización Wnt/fisiología , Lesión Renal Aguda/fisiopatología , Animales , Progresión de la Enfermedad , Fibrosis , Humanos , Riñón/patología , Túbulos Renales/patología , Túbulos Renales/fisiopatología , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
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.
Asunto(s)
Proteína Forkhead Box O3/metabolismo , Túbulos Renales Proximales/metabolismo , Insuficiencia Renal Crónica/metabolismo , Transducción de Señal , beta Catenina/metabolismo , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Proteína Forkhead Box O3/genética , Túbulos Renales Proximales/patología , Masculino , Ratones , Ratones Transgénicos , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , beta Catenina/genéticaRESUMEN
Transforming growth factor-ß (TGF-ß) is a key profibrotic growth factor that is activated in acute kidney injury (AKI) and associated with cellular responses that lead to the development of chronic kidney disease (CKD). The persistently injured, de-differentiated tubular epithelial cell is an important mediator of the transition from AKI to CKD. TGF-ß signaling may perpetuate proximal tubule injury through de-differentiation, cell cycle arrest, and increased susceptibility to apoptosis. In addition, TGF-ß signaling promotes macrophage chemotaxis, endothelial injury, and myofibroblast differentiation after AKI. Future studies that block TGF-ß signaling after cessation of AKI are needed to better define its role in the progression of acute to chronic renal injury.
Asunto(s)
Lesión Renal Aguda/patología , Insuficiencia Renal Crónica/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Progresión de la Enfermedad , Humanos , Transducción de SeñalAsunto(s)
Envejecimiento , Carnitina O-Palmitoiltransferasa , Insuficiencia Renal Crónica , Envejecimiento/genética , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/deficiencia , Túbulos Renales/patología , Túbulos Renales/metabolismo , Insuficiencia Renal Crónica/genéticaRESUMEN
Tubulointerstitial fibrosis (TIF) is the hallmark of chronic kidney disease and best predictor of renal survival. Many different cell types contribute to TIF progression including tubular epithelial cells, myofibroblasts, endothelia, and inflammatory cells. Previously, most of the attention has centered on myofibroblasts given their central importance in extracellular matrix production. However, emerging data focuses on how the response of the proximal tubule, a specialized epithelial segment vulnerable to injury, plays a central role in TIF progression. Several proximal tubular responses such as de-differentiation, cell cycle changes, autophagy, and metabolic changes may be adaptive initially, but can lead to maladaptive responses that promote TIF both through autocrine and paracrine effects. This review discusses the current paradigm of TIF progression and the increasingly important role of the proximal tubule in promoting TIF both in tubulointerstitial and glomerular injuries. A better understanding and appreciation of the role of the proximal tubule in TIF has important implications for therapeutic strategies to halt chronic kidney disease progression.
Asunto(s)
Túbulos Renales Proximales/patología , Insuficiencia Renal Crónica/patología , Animales , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Fibrosis , Humanos , Túbulos Renales Proximales/metabolismo , Macrófagos/metabolismo , Miofibroblastos/metabolismo , Insuficiencia Renal Crónica/metabolismoRESUMEN
Wnt/ß-catenin signaling is extremely important for proper kidney development. This pathway is also upregulated in injured renal tubular epithelia, both in acute kidney injury and chronic kidney disease. The renal tubular epithelium is an important target of kidney injury, and its response (repair versus persistent injury) is critical for determining whether tubulointerstitial fibrosis, the hallmark of chronic kidney disease, develops. This review discusses how Wnt/ß-catenin signaling in the injured tubular epithelia promotes either repair or fibrosis after kidney injury. There is data suggesting that epithelial Wnt/ß-catenin signaling is beneficial in acute kidney injury and important in tubular progenitors responsible for epithelial repair. The role of Wnt/ß-catenin signaling in chronically injured epithelia is less clear. There is convincing data that Wnt/ß-catenin signaling in interstitial fibroblasts and pericytes contributes to the extracellular matrix accumulation that defines fibrosis. However, some recent studies question whether Wnt/ß-catenin signaling in chronically injured epithelia actually promotes fibrosis or repair.