RESUMO
Induced pluripotent stem cells (iPSC) have huge potential as cell therapy for various diseases, given their potential for unlimited self-renewal and capability to differentiate into a wide range of cell types. Although autologous iPSCs represents the ideal source for patient-tailored regenerative medicine, the high costs of the extensive and time-consuming production process and the impracticability for treating acute conditions hinder their use for broad applications. An allogeneic iPSC-based strategy may overcome these issues, but it carries the risk of triggering an immune response. So far, several approaches based on genome-editing techniques to silence human leukocyte antigen class I (HLA-I) or II (HLA-II) expression have been explored to overcome the immune rejection of allogeneic iPSCs. In this study, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the ß2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes, essential for the correct surface expression of HLA-I and HLA-II proteins. The resulting hypoimmunogenic iPSC line has a normal karyotype, expresses the pluripotency stem cell markers, and is capable of differentiating into the three embryonic germ layers. Furthermore, we showed that it specifically retains the ability to differentiate towards different liver cells, such as endothelial-like cells, hepatocyte-like cells, and hepatic stellate-like cells. Our results indicate that hypoimmunogenic iPSCs could give a new cost-effective and off-the-shelf opportunity for cell therapy in liver diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Medicina Regenerativa , Edição de Genes/métodos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , FígadoRESUMO
No effective treatments are available for familial steroid-resistant Focal Segmental Glomerulosclerosis (FSGS), characterized by proteinuria due to ultrastructural abnormalities in glomerular podocytes. Here, we studied a private PAX2 mutation identified in a patient who developed FSGS in adulthood. By generating adult podocytes using patient-specific induced pluripotent stem cells (iPSC), we developed an in vitro model to dissect the role of this mutation in the onset of FSGS. Despite the PAX2 mutation, patient iPSC properly differentiated into podocytes that exhibited a normal structure and function when compared to control podocytes. However, when exposed to an environmental trigger, patient podocytes were less viable and more susceptible to cell injury. Fixing the mutation improved their phenotype and functionality. Using a branching morphogenesis assay, we documented developmental defects in patient-derived ureteric bud-like tubules that were totally rescued by fixing the mutation. These data strongly support the hypothesis that the PAX2 mutation has a dual effect, first in renal organogenesis, which could account for a suboptimal nephron number at birth, and second in adult podocytes, which are more susceptible to cell death caused by environmental triggers. These abnormalities might translate into the development of proteinuria in vivo, with a progressive decline in renal function, leading to FSGS.
RESUMO
Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the "Nichoid", an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or "2D Nichoid" and multi-layered or "3D Nichoid") were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells' specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells' features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo.
Assuntos
Células-Tronco Mesenquimais/citologia , Animais , Western Blotting , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Imunofluorescência , Adesões Focais/fisiologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Road traffic is one of the main sources of particulate emissions into the environment and has an increasing, negative impact on the release of potentially dangerous materials. Vehicle brakes release a significant amount of wear particles, and knowledge regarding their possible adverse effects is limited. One of the most dangerous elements contained in brake pads is copper (Cu), known to be toxic for human health. Therefore, our aim was to study the cell toxicity of particulate matter (PM) produced by different combinations of braking discs and pads containing different amounts of Cu. We investigated whether brake-derived microparticles have toxic effects on lung cells proportionally to their Cu content. Analyte content was measured in friction materials by XRFS and in PM2.5 captured during braking tests using SEM/EDX. The biological impact of brake-derived PM2.5 was investigated on a human epithelial alveolar cell line (A549). Cell viability, oxidative stress, mitochondrial membrane potential, apoptosis, and the pro-inflammatory response of the cells, as well as gene expression, were assessed following exposure to increasing PM2.5 concentrations (1, 10, 100, 200, and 500 µg/ml). The brake debris with the lowest Cu content did not induce significant changes in biological effects on A549 cells compared to normal controls, except for ROS production and IL6 gene expression. PM2.5 containing higher Cu quantities induced cell toxicity that correlated with Cu concentration. Our data suggest that the toxicity of PM2.5 from the brake system is mainly related to Cu content, thus confirming that eliminating Cu from brake pads will be beneficial for human health in urbanized environments.
Assuntos
Poluentes Atmosféricos/toxicidade , Cobre/toxicidade , Material Particulado/toxicidade , Células Epiteliais Alveolares/efeitos dos fármacos , Humanos , Estresse Oxidativo , Emissões de VeículosRESUMO
Focal segmental glomerulosclerosis (FSGS) is defined by focal (involving few glomeruli) and segmental sclerosis of the glomerular tuft that manifests with nephrotic syndrome. Mutations in genes involved in the maintenance of structure and function of podocytes have been found in a minority of these patients. A family with adult-onset autosomal dominant FSGS was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2. Here, we efficiently corrected this point mutation in patient-derived induced pluripotent stem cells (iPSCs) by means of CRISPR-Cas9-based homology-directed repair. The iPSC lines were differentiated into podocytes, which were tested for their motility. Editing the PAX2 p.G189R mutation restored podocyte motility, which was altered in podocytes derived from patient iPSCs.
Assuntos
Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/terapia , Fator de Transcrição PAX2/genética , Adulto , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Engenharia Genética/métodos , Mutação em Linhagem Germinativa/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Glomérulos Renais/metabolismo , Mutação/genética , Fator de Transcrição PAX2/análise , Podócitos/química , Podócitos/metabolismo , Podócitos/fisiologia , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Mesenchymal stromal cells (MSCs) are renoprotective and drive regeneration following injury, although cellular targets of such an effect are still ill-defined. Here, we show that human umbilical cord (UC)-MSCs transplanted into mice stimulate tubular cells to regain mitochondrial mass and function, associated with enhanced microtubule-rich projections that appear to mediate mitochondrial trafficking to create a reparative dialogue among adjacent tubular cells. Treatment with UC-MSCs in mice with cisplatin-induced acute kidney injury (AKI) regulates mitochondrial biogenesis in proximal tubuli by enhancing PGC1α expression, NAD+ biosynthesis and Sirtuin 3 (SIRT3) activity, thus fostering antioxidant defenses and ATP production. The functional role of SIRT3 in tubular recovery is highlighted by data that in SIRT3-deficient mice with AKI, UC-MSC treatment fails to induce renoprotection. These data document a previously unrecognized mechanism through which UC-MSCs facilitate renal repair, so as to induce global metabolic reprogramming of damaged tubular cells to sustain energy supply.Mesenchymal stromal cells drive renal regeneration following injury. Here, the authors show that human mesenchymal stromal cells, when transplanted into mice with acute kidney injury, stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3.
Assuntos
Injúria Renal Aguda/terapia , Túbulos Renais/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Trifosfato de Adenosina/metabolismo , Animais , Proliferação de Células , Cisplatino/efeitos adversos , Feminino , Humanos , Camundongos , Camundongos SCID , Mitocôndrias/genética , NAD/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
We have previously shown that rat allogeneic DC, made immature by adenoviral gene transfer of the dominant negative form of IKK2, gave rise in-vitro to a unique population of CD4+CD25- regulatory T cells (dnIKK2-Treg). These cells inhibited Tcell response in-vitro, without needing cell-to-cell contact, and induced kidney allograft survival prolongation in-vivo. Deep insight into the mechanisms behind dnIKK2-Treg-induced suppression of Tcell proliferation remained elusive. Here we document that dnIKK2-Treg release extracellular vesicles (EV) riched in exosomes, fully accounting for the cell-contact independent immunosuppressive activity of parent cells. DnIKK2-Treg-EV contain a unique molecular cargo of specific miRNAs and iNOS, which, once delivered into target cells, blocked cell cycle progression and induced apoptosis. DnIKK2-Treg-EV-exposed T cells were in turn converted into regulatory cells. Notably, when administered in-vivo, dnIKK2-Treg-EV prolonged kidney allograft survival. DnIKK2-Treg-derived EV could be a tool for manipulating the immune system and for discovering novel potential immunosuppressive molecules in the context of allotransplantation.
Assuntos
Aloenxertos/fisiologia , Vesículas Extracelulares/metabolismo , Tolerância Imunológica , Imunossupressores/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , RatosRESUMO
Dabrafenib and trametinib, BRAF and MEK inhibitors, respectively, are effective targeted metastatic melanoma therapies, but little is known about their nephrotoxicity. Although tubulointerstitial injury has been the most widely reported renal side effect of targeted melanoma therapy, nephrotic syndrome has not been reported before. We report on a patient with metastatic melanoma who developed nephrotic syndrome during dabrafenib and trametinib treatment. Kidney biopsy showed diffuse loss of podocyte cytoarchitecture, extensive foot-process effacement, and glomerular endothelial injury. Kidney function and glomerular ultrastructural changes recovered fully after drug withdrawal. In vitro, BRAF inhibition decreased PLCε1 expression in podocytes, accompanied by a reduction in nephrin expression and an increase in permeability to albumin. Additionally, these drugs inhibited the podocyte-vascular endothelial growth factor (VEGF) system. In addition to implications for nephrotic syndrome pathophysiology, we suggest that patients given dabrafenib and trametinib be monitored closely for potential glomerular damage.
Assuntos
Antineoplásicos/efeitos adversos , Imidazóis/efeitos adversos , Melanoma/tratamento farmacológico , Síndrome Nefrótica/induzido quimicamente , Oximas/efeitos adversos , Podócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Piridonas/efeitos adversos , Pirimidinonas/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Feminino , Humanos , Proteínas Proto-Oncogênicas B-raf/fisiologiaRESUMO
Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Podócitos/citologia , Actinina/genética , Actinina/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Células Cultivadas , Corpos Embrioides/metabolismo , Corpos Embrioides/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Cariótipo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Podócitos/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMO
Podocyte loss is the initial event in the development of glomerulosclerosis, the structural hallmark of progressive proteinuric nephropathies. Understanding mechanisms underlying glomerular injury is the key challenge for identifying novel therapeutic targets. In mice with protein-overload induced by bovine serum albumin (BSA), we evaluated whether the alternative pathway (AP) of complement mediated podocyte depletion and podocyte-dependent parietal epithelial cell (PEC) activation causing glomerulosclerosis. Factor H (Cfh(-/-)) or factor B-deficient mice were studied in comparison with wild-type (WT) littermates. WT+BSA mice showed podocyte depletion accompanied by glomerular complement C3 and C3a deposits, PEC migration to capillary tuft, proliferation, and glomerulosclerosis. These changes were more prominent in Cfh(-/-) +BSA mice. The pathogenic role of AP was documented by data that factor B deficiency preserved glomerular integrity. In protein-overload mice, PEC dysregulation was associated with upregulation of CXCR4 and GDNF/c-Ret axis. In vitro studies provided additional evidence of a direct action of C3a on proliferation and CXCR4-related migration of PECs. These effects were enhanced by podocyte-derived GDNF. In patients with proteinuric nephropathy, glomerular C3/C3a paralleled PEC activation, CXCR4 and GDNF upregulation. These results indicate that mechanistically uncontrolled AP complement activation is not dispensable for podocyte-dependent PEC activation resulting in glomerulosclerosis.
Assuntos
Complemento C3a/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Adulto , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Fator B do Complemento/deficiência , Fator B do Complemento/genética , Fator H do Complemento/deficiência , Fator H do Complemento/genética , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Podócitos/citologia , Podócitos/metabolismo , Proteinúria/etiologia , Soroalbumina Bovina/administração & dosagem , Regulação para Cima , Adulto JovemRESUMO
Chronic renal insufficiency inexorably progresses in patients, such as it does after partial renal ablation in rats. However, the progression of renal diseases can be delayed by angiotensin II blockers that stabilize renal function or increase GFR, even in advanced phases of the disease. Regression of glomerulosclerosis can be induced by angiotensin II antagonism, but the effect of these treatments on the entire vascular tree is unclear. Here, using microcomputed tomography and scanning electron microscopy, we compared the size and extension of kidney blood vessels in untreated Wistar rats with those in untreated and angiotensin II antagonist-treated Munich Wistar Frömter (MWF) rats that spontaneously develop kidney disease with age. The kidney vasculature underwent progressive rarefaction in untreated MWF rats, substantially affecting intermediate and small vessels. Microarray analysis showed increased Tgf-ß and endothelin-1 gene expression with age. Notably, 10-week inhibition of the renin-angiotensin system regenerated kidney vasculature and normalized Tgf-ß and endothelin-1 gene expression in aged MWF rats. These changes were associated with reduced apoptosis, increased endothelial cell proliferation, and restoration of Nrf2 expression, suggesting mechanisms by which angiotensin II antagonism mediates regeneration of capillary segments. These results have important implications in the clinical setting of chronic renal insufficiency.
Assuntos
Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Capilares/fisiologia , Glomérulos Renais/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Insuficiência Renal Crônica/tratamento farmacológico , Actinas/metabolismo , Animais , Apoptose , Capilares/metabolismo , Capilares/ultraestrutura , Proliferação de Células , Células Endoteliais/fisiologia , Endotelina-1/genética , Expressão Gênica , Microscopia Eletrônica de Varredura , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Wistar , Sistema Renina-Angiotensina/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Microtomografia por Raio-XRESUMO
Intimal hyperplasia (IH) is the first cause of failure of an arteriovenous fistula (AVF). The aim of the present study was to investigate the effects on endothelial cells (ECs) of shear stress waveforms derived from AVF areas prone to develop IH. We used a cone-and-plate device to obtain real-time control of shear stress acting on EC cultures. We exposed human umbilical vein ECs for 48 h to different shear stimulations calculated in a side-to-end AVF model. Pulsatile unidirectional flow, representative of low-risk stenosis areas, induced alignment of ECs and actin fiber orientation with flow. Shear stress patterns of reciprocating flow, derived from high-risk stenosis areas, did not affect EC shape or cytoskeleton organization, which remained similar to static cultures. We also evaluated flow-induced EC expression of genes known to be involved in cytoskeletal remodeling and expression of cell adhesion molecules. Unidirectional flow induced a significant increase in Kruppel-like factor 2 mRNA expression, whereas it significantly reduced phospholipase D1, α4-integrin, and Ras p21 protein activator 1 mRNA expression. Reciprocating flow did not increase Kruppel-like factor 2 mRNA expression compared with static controls but significantly increased mRNA expression of phospholipase D1, α4-integrin, and Ras p21 protein activator 1. Reciprocating flow selectively increased monocyte chemoattractant protein-1 and IL-8 production. Furthermore, culture medium conditioned by ECs exposed to reciprocating flows selectively increased smooth muscle cell proliferation compared with unidirectional flow. Our results indicate that protective vascular effects induced in ECs by unidirectional pulsatile flow are not induced by reciprocating shear forces, suggesting a mechanism by which oscillating flow conditions may induce the development of IH in AVF and vascular access dysfunction.
Assuntos
Derivação Arteriovenosa Cirúrgica/efeitos adversos , Hemodinâmica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mecanotransdução Celular , Diálise Renal , Citoesqueleto de Actina/metabolismo , Proliferação de Células , Forma Celular , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Hiperplasia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Comunicação Parácrina , Fluxo Pulsátil , RNA Mensageiro/metabolismo , Transdução de Sinais , Estresse Mecânico , Fatores de TempoRESUMO
BACKGROUND: Angiotensin II promotes insulin resistance. The mechanism underlying this abnormality, however, is still poorly defined. In a different setting, skeletal muscle metabolism and insulin signaling are regulated by Sirtuin3. OBJECTIVE: Here, we investigate whether angiotensin II-induced insulin resistance in skeletal muscle is associated with Sirtuin3 dysregulation and whether pharmacological manipulation of Sirtuin3 confers protection. STUDY DESIGN: Parental and GLUT4-myc L6 rat skeletal muscle cells exposed to angiotensin II are used as in vitro models of insulin resistance. GLUT4 translocation, glucose uptake, intracellular molecular signals such as mitochondrial reactive oxygen species, Sirtuin3 protein expression and activity, along with its downstream targets and upstream regulators, are analyzed both in the absence and presence of acetyl-L-carnitine. The role of Sirtuin3 in GLUT4 translocation and intracellular molecular signaling is also studied in Sirtuin3-silenced as well as over-expressing cells. RESULTS: Angiotensin II promotes insulin resistance in skeletal muscle cells via mitochondrial oxidative stress, resulting in a two-fold increase in superoxide generation. In this context, reactive oxygen species open the mitochondrial permeability transition pore and significantly lower Sirtuin3 levels and activity impairing the cell antioxidant defense. Angiotensin II-induced Sirtuin3 dysfunction leads to the impairment of AMP-activated protein kinase/nicotinamide phosphoribosyltransferase signaling. Acetyl-L-carnitine, by lowering angiotensin II-induced mitochondrial superoxide formation, prevents Sirtuin3 dysfunction. This phenomenon implies the restoration of manganese superoxide dismutase antioxidant activity and AMP-activated protein kinase activation. Acetyl-L-carnitine protection is abrogated by specific Sirtuin3 siRNA. CONCLUSIONS: Our data demonstrate that angiotensin II-induced insulin resistance fosters mitochondrial superoxide generation, in turn leading to Sirtuin3 dysfunction. The present results also highlight Sirtuin3 as a therapeutic target for the insulin-sensitizing effects of acetyl-L-carnitine.
Assuntos
Angiotensina II/farmacologia , Resistência à Insulina , Músculo Esquelético/fisiologia , Sirtuína 3/fisiologia , Acetilcarnitina/farmacologia , Animais , Linhagem Celular , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Músculo Esquelético/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.
Assuntos
Diferenciação Celular , Reprogramação Celular , Rim/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Linhagem Celular Transformada , Sistema Livre de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Túbulos Renais Proximais/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , TranscriptomaRESUMO
New intervention tools for severely damaged kidneys are in great demand to provide patients with a valid alternative to whole organ replacement. For repairing or replacing injured tissues, emerging approaches focus on using stem and progenitor cells. Embryonic kidneys represent an interesting option because, when transplanted to sites such as the renal capsule of healthy animals, they originate new renal structures. Here, we studied whether metanephroi possess developmental capacity when transplanted under the kidney capsule of MWF male rats, a model of spontaneous nephropathy. We found that six weeks post-transplantation, renal primordia developed glomeruli and tubuli able to filter blood and to produce urine in cyst-like structures. Newly developed metanephroi were able to initiate a regenerative-like process in host renal tissues adjacent to the graft in MWF male rats as indicated by an increase in cell proliferation and vascular density, accompanied by mRNA and protein upregulation of VEGF, FGF2, HGF, IGF-1 and Pax-2. The expression of SMP30 and NCAM was induced in tubular cells. Oxidative stress and apoptosis markedly decreased. Our study shows that embryonic kidneys generate functional nephrons when transplanted into animals with severe renal disease and at the same time activate events at least partly mimicking those observed in kidney tissues during renal regeneration.
Assuntos
Nefropatias/terapia , Transplante de Rim , Regeneração , Animais , Apoptose , Biomarcadores , Proliferação de Células , Feminino , Fibroblastos/metabolismo , Fibroblastos/transplante , Expressão Gênica , Sobrevivência de Enxerto , Nefropatias/patologia , Nefropatias/fisiopatologia , Transplante de Rim/métodos , Masculino , Neovascularização Fisiológica , Estresse Oxidativo , Ratos , Regeneração/genéticaRESUMO
Acute kidney injury (AKI) is a public health concern with an annual mortality rate that exceeds those of breast and prostate cancer, heart failure, and diabetes combined. Oxidative stress and mitochondrial damage are drivers of AKI-associated pathology; however, the pathways that mediate these events are poorly defined. Here, using a murine cisplatin-induced AKI model, we determined that both oxidative stress and mitochondrial damage are associated with reduced levels of renal sirtuin 3 (SIRT3). Treatment with the AMPK agonist AICAR or the antioxidant agent acetyl-l-carnitine (ALCAR) restored SIRT3 expression and activity, improved renal function, and decreased tubular injury in WT animals, but had no effect in Sirt3-/- mice. Moreover, Sirt3-deficient mice given cisplatin experienced more severe AKI than WT animals and died, and neither AICAR nor ALCAR treatment prevented death in Sirt3-/- AKI mice. In cultured human tubular cells, cisplatin reduced SIRT3, resulting in mitochondrial fragmentation, while restoration of SIRT3 with AICAR and ALCAR improved cisplatin-induced mitochondrial dysfunction. Together, our results indicate that SIRT3 is protective against AKI and suggest that enhancing SIRT3 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury.
Assuntos
Injúria Renal Aguda/enzimologia , Túbulos Renais Proximais/enzimologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Sirtuína 3/metabolismo , Acetilcarnitina/farmacologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , Cisplatino/farmacocinética , Humanos , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Sirtuína 3/genética , Complexo Vitamínico B/farmacologiaRESUMO
Activation of endothelin-A receptor (ET(A)R) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through ß-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker α-smooth muscle actin. ET-1 promoted podocyte migration via ET(A)R activation and increased ß-arrestin-1 expression. Activated ET(A)R recruited ß-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and ß-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1-induced migration as confirmed by Snail knockdown experiments. Silencing of ß-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ET(A)R-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; ß-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ET(A)R antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular ß-arrestin-1 and Snail. Increased ß-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ET(A)R antagonism for a group of diseases still needing a specific treatment.
Assuntos
Arrestinas/fisiologia , Endotelina-1/metabolismo , Glomerulonefrite/induzido quimicamente , Podócitos/fisiologia , Receptor de Endotelina A/metabolismo , Animais , Movimento Celular , Modelos Animais de Doenças , Doxorrubicina , Receptores ErbB/metabolismo , Feminino , Glomerulonefrite/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Ativação Transcricional , beta Catenina/metabolismo , beta-Arrestina 1 , beta-Arrestinas , Quinases da Família src/metabolismoRESUMO
In nondiabetic rat models of renal disease, angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. Herein, we wanted to explore the role of Ang II in diabetic nephropathy by a translational approach spanning from in vitro to in vivo rat and human studies, and to dissect the intracellular pathways involved. In isolated perfused rat kidneys and in cultured human podocytes, Ang II down-regulated nephrin expression via Notch1 activation and nuclear translocation of Snail. Hairy enhancer of split-1 was a Notch1-downstream gene effector that activated Snail in cultured podocytes. In vitro changes of the Snail/nephrin axis were similar to those in renal biopsy specimens of Zucker diabetic fatty rats and patients with advanced diabetic nephropathy, and were normalized by pharmacological inhibition of the renin-angiotensin system. Collectively, the present studies provide evidence that Ang II plays a relevant role in perpetuating glomerular injury in experimental and human diabetic nephropathy via persistent activation of Notch1 and Snail signaling in podocytes, eventually resulting in down-regulation of nephrin expression, the integrity of which is crucial for the glomerular filtration barrier.
Assuntos
Angiotensina II/metabolismo , Nefropatias Diabéticas/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Regulação para Baixo , Feminino , Humanos , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Modelos Lineares , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição da Família SnailRESUMO
Bone marrow-mesenchymal stem cells (BM-MSC) ameliorate renal dysfunction and repair tubular damage of acute kidney injury by locally releasing growth factors, including the insulin-like growth factor-1 (IGF-1). The restricted homing of BM-MSC at the site of injury led us to investigate a possible gene-based communication mechanism between BM-MSC and tubular cells. Human BM-MSC (hBM-MSC) released microparticles and exosomes (Exo) enriched in mRNAs. A selected pattern of transcripts was detected in Exo versus parental cells. Exo expressed the IGF-1 receptor (IGF-1R), but not IGF-1 mRNA, while hBM-MSC contained both mRNAs. R- cells lacking IGF-1R exposed to hBM-MSC-derived Exo acquired the human IGF-1R transcript that was translated in the corresponding protein. Transfer of IGF-1R mRNA from Exo to cisplatin-damaged proximal tubular cells (proximal tubular epithelial cell [PTEC]) increased PTEC proliferation. Coincubation of damaged PTEC with Exo and soluble IGF-1 further enhanced cell proliferation. These findings suggest that horizontal transfer of the mRNA for IGF-1R to tubular cells through Exo potentiates tubular cell sensitivity to locally produced IGF-1 providing a new mechanism underlying the powerful renoprotection of few BM-MSC observed in vivo.
Assuntos
Exossomos , Transferência Genética Horizontal , Células-Tronco Mesenquimais/fisiologia , RNA Mensageiro/genética , Receptores de Fatores de Crescimento/genética , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Adulto , Animais , Células da Medula Óssea/metabolismo , Comunicação Celular , Proliferação de Células , Células Cultivadas , Humanos , Fator de Crescimento Insulin-Like I/genética , Túbulos Renais Proximais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente PequenoRESUMO
In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)-MSCs to cure mice with AKI. Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1 beta and TNFalpha synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.