RESUMO
Background: Our laboratory has previously demonstrated that Sirt1endo-/- mice show endothelial dysfunction and exaggerated renal fibrosis, whereas mice with silenced endothelial transforming growth factor beta (TGF-ß) signaling are resistant to fibrogenic signals. Considering the fact that the only difference between these mutant mice is confined to the vascular endothelium, this indicates that secreted substances contribute to these contrasting responses. Methods: We performed an unbiased proteomic analysis of the secretome of renal microvascular endothelial cells (RMVECs) isolated from these two mutants. We cultured renal fibroblasts and RMVECs and used microfluidic devices for coculturing. Results: Dickkopf-3 (DKK3), a putative ligand of the Wnt/ß-catenin pathway, was present exclusively in the fibrogenic secretome. In cultured fibroblasts, DKK3 potently induced myofibroblast activation. In addition, DKK3 antagonized effects of DKK1, a known inhibitor of the Wnt pathway, in conversion of fibroblasts to myofibroblasts. In RMVECs, DKK3 induced endothelial-mesenchymal transition and impaired their angiogenic competence. The inhibition of endothelial outgrowth, enhanced myofibroblast formation and endothelial-mesenchymal transition were confirmed in coculture. In reporter DKK3-eGFP × Col3.6-GFPcyan mice, DKK3 was marginally expressed under basal conditions. Adriamycin-induced nephropathy resulted in upregulation of DKK3 expression in tubular and, to a lesser degree, endothelial compartments. Sulindac sulfide was found to exhibit superior Wnt pathway-suppressive action and decreased DKK3 signals and the extent of renal fibrosis. Conclusions: In conclusion, this unbiased proteomic screen of the profibrogenic endothelial secretome revealed DKK3 acting as an agonist of the Wnt pathway, enhancing formation of myofibroblasts and endothelial-mesenchymal transition and impairing angiogenesis. A potent inhibitor of the Wnt pathway, sulindac sulfide, suppressed nephropathy-induced DKK3 expression and renal fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Endotélio Vascular/patologia , Transição Epitelial-Mesenquimal , Fibrose/patologia , Nefropatias/patologia , Proteoma/análise , Receptor do Fator de Crescimento Transformador beta Tipo II/fisiologia , Sirtuína 1/fisiologia , Animais , Endotélio Vascular/metabolismo , Fibrose/metabolismo , Nefropatias/metabolismo , Camundongos , Camundongos Knockout , Proteômica , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
During sepsis, the alarmin HMGB1 is released from tissues and promotes systemic inflammation that results in multi-organ damage, with the kidney particularly susceptible to injury. The severity of inflammation and pro-damage signaling mediated by HMGB1 appears to be dependent on the alarmin's redox state. Therefore, we examined HMGB1 redox in kidney cells during sepsis. Using intravital microscopy, CellROX labeling of kidneys in live mice indicated increased ROS generation in the kidney perivascular endothelium and tubules during lipopolysaccharide (LPS)-induced sepsis. Subsequent CellROX and MitoSOX labeling of LPS-stressed endothelial and kidney proximal tubule cells demonstrated increased ROS generation in these cells as sepsis worsens. Consequently, HMGB1 oxidation increased in the cytoplasm of kidney cells during its translocation from the nucleus to the circulation, with the degree of oxidation dependent on the severity of sepsis, as measured in in vivo mouse samples using a thiol assay and mass spectrometry (LC-MS/MS). The greater the oxidation of HMGB1, the greater the ability of the alarmin to stimulate pro-inflammatory cyto-/chemokine release (measured by Luminex Multiplex) and alter mitochondrial ATP generation (Luminescent ATP Detection Assay). Administration of glutathione and thioredoxin inhibitors to cell cultures enhanced HMGB1 oxidation during sepsis in endothelial and proximal tubule cells, respectively. In conclusion, as sepsis worsens, ROS generation and HMGB1 oxidation increases in kidney cells, which enhances HMGB1's pro-inflammatory signaling. Conversely, the glutathione and thioredoxin systems work to maintain the protein in its reduced state.
Assuntos
Proteína HMGB1/metabolismo , Estresse Oxidativo , Sepse/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Citocinas/metabolismo , Proteína HMGB1/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Accumulation of myofibroblasts is a hallmark of renal fibrosis. A significant proportion of myofibroblasts has been reported to originate via endothelial-mesenchymal transition. We initially hypothesized that exposing myofibroblasts to the extract of endothelial progenitor cells (EPCs) could reverse this transition. Indeed, in vitro treatment of transforming growth factor-ß1 (TGF-ß1)-activated fibroblasts with EPC extract prevented expression of α-smooth muscle actin (α-SMA); however, it did not enhance expression of endothelial markers. In two distinct models of renal fibrosis-unilateral ureteral obstruction and chronic phase of folic acid-induced nephropathy-subcapsular injection of EPC extract to the kidney prevented and reversed accumulation of α-SMA-positive myofibroblasts and reduced fibrosis. Screening the composition of EPC extract for cytokines revealed that it is enriched in leukemia inhibitory factor (LIF) and vascular endothelial growth factor. Only LIF was capable of reducing fibroblast-to-myofibroblast transition of TGF-ß1-activated fibroblasts. In vivo subcapsular administration of LIF reduced the number of myofibroblasts and improved the density of peritubular capillaries; however, it did not reduce the degree of fibrosis. A receptor-independent ligand for the gp130/STAT3 pathway, hyper-interleukin-6 (hyper-IL-6), not only induced a robust downstream increase in pluripotency factors Nanog and c-Myc but also exhibited a powerful antifibrotic effect. In conclusion, EPC extract prevented and reversed fibroblast-to-myofibroblast transition and renal fibrosis. The component of EPC extract, LIF, was capable of preventing development of the contractile phenotype of activated fibroblasts but did not eliminate TGF-ß1-induced collagen synthesis in cultured fibroblasts and models of renal fibrosis, whereas a receptor-independent gp130/STAT3 agonist, hyper-IL-6, prevented fibrosis. In summary, these studies, through the evolution from EPC extract to LIF and then to hyper-IL-6, demonstrate the instructive role of microenvironmental cues and may provide in the future a facile strategy to prevent and reverse renal fibrosis. Stem Cells Translational Medicine 2017;6:992-1005.
Assuntos
Microambiente Celular , Rim/patologia , Células 3T3 , Animais , Microambiente Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Receptor gp130 de Citocina/metabolismo , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibrose , Interleucina-6/farmacologia , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/metabolismo , Obstrução Ureteral/patologiaRESUMO
We recently demonstrated the use of in vitro expanded kidney-derived mesenchymal stem cells (KMSC) protected peritubular capillary endothelial cells in acute renal ischemia-reperfusion injury. Herein, we isolated and characterized microparticles (MPs) from KMSC. We investigated their in vitro biologic effects on human endothelial cells and in vivo renoprotective effects in acute ischemia-reperfusion renal injury. MPs were isolated from the supernatants of KMSC cultured in anoxic conditions in serum-deprived media for 24 hours. KMSC-derived MPs demonstrated the presence of several adhesion molecules normally expressed on KMSC membranes, such as CD29, CD44, CD73, α4, 5, and 6 integrins. Quantitative real time PCR confirmed the presence of 3 splicing variants of VEGF-A (120, 164, 188), bFGF and IGF-1 in isolated MPs. MPs labeled with PKH26 red fluorescence dye were incorporated by cultured human umbilical vein endothelial cells (HUVEC) via surface molecules such as CD44, CD29, and α4, 5, and 6 integrins. MP dose dependently improved in vitro HUVEC proliferation and promoted endothelial tube formation on growth factor reduced Matrigel. Moreover, apoptosis of human microvascular endothelial cell was inhibited by MPs. Administration of KMSC-derived MPs into mice with acute renal ischemia was followed by selective engraftment in ischemic kidneys and significant improvement in renal function. This was achieved by improving proliferation, of peritubular capillary endothelial cell and amelioration of peritubular microvascular rarefaction. Our results support the hypothesis that KMSC-derived MPs may act as a source of proangiogenic signals and confer renoprotective effects in ischemic kidneys.
Assuntos
Injúria Renal Aguda/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Apoptose , Transporte Biológico , Proliferação de Células , Micropartículas Derivadas de Células/ultraestrutura , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Transferência Genética Horizontal , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Neovascularização Fisiológica , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
Factors that initiate cellular damage and trigger the inflammatory response cascade and renal injury are not completely understood after renal ischemia-reperfusion injury (IRI). High-mobility group box-1 protein (HMGB1) is a damage-associated molecular pattern molecule that binds to chromatin, but upon signaling undergoes nuclear-cytoplasmic translocation and release from cells. Immunohistochemical and Western blot analysis identified HMGB1 nuclear-cytoplasmic translocation and release from renal cells (particularly vascular and tubular cells) into the venous circulation after IRI. Time course analysis indicated HMGB1 release into the venous circulation progressively increased parallel to increased renal ischemic duration. Ethyl pyruvate (EP) treatment blocked H(2)O(2) (oxidative stress)-induced HMGB1 release from human umbilical vein endothelial cells in vitro, and in vivo resulted in nuclear retention and significant blunting of HMGB1 release into the circulation after IRI. EP treatment before IRI improved short-term serum creatinine and albuminuria, proinflammatory cyto-/chemokine release, and long-term albuminuria and fibrosis. The renoprotective effect of EP was abolished when exogenous HMGB1 was injected, suggesting EP's therapeutic efficacy is mediated by blocking HMGB1 translocation and release. To determine the independent effects of circulating HMGB1 after injury, exogenous HMGB1 was administered to healthy animals at pathophysiological dose. HMGB1 administration induced a rapid surge in systemic circulating cyto-/chemokines (including TNF-α, eotaxin, G-CSF, IFN-γ, IL-10, IL-1α, IL-6, IP-10, and KC) and led to mobilization of bone marrow CD34+Flk1+ cells into the circulation. Our results indicate that increased ischemic duration causes progressively enhanced HMGB1 release into the circulation triggering damage/repair signaling, an effect inhibited by EP because of its ability to block HMGB1 nuclear-cytoplasmic translocation.
Assuntos
Proteínas de Grupo de Alta Mobilidade/metabolismo , Rim/irrigação sanguínea , Traumatismo por Reperfusão/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células da Medula Óssea/fisiologia , Creatinina/sangue , Citocinas/sangue , Proteínas de Grupo de Alta Mobilidade/sangue , Proteínas de Grupo de Alta Mobilidade/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Piruvatos/farmacologia , Proteínas Repressoras/sangue , Proteínas Repressoras/farmacologiaRESUMO
Sepsis and its complications are associated with poor clinical outcomes. The circulatory system is a well-known target of lipopolysaccharide (LPS). Recently, several clinical studies documented mobilization of endothelial progenitor cells (EPCs) during endotoxemia, with the probability of patients' survival correlating with the rise in circulating EPCs. This fact combined with endotoxemia-induced vascular injury led us to hypothesize that the developing functional EPC incompetence could impede vascular repair and that adoptive transfer of EPCs could improve hemodynamics in endotoxemia. We used LPS injection to model endotoxemia. EPCs isolated from endotoxemic mice exhibited impaired clonogenic potential and LPS exerted Toll-like receptor 4-mediated cytotoxic effects toward EPCs, which was mitigated by embedding them in hyaluronic acid (HA) hydrogels. Therefore, intact EPCs were either delivered intravenously or embedded within pronectin-coated HA hydrogels. Adoptive transfer of EPCs in LPS-injected mice improved control of blood pressure and reduced hepatocellular and renal dysfunction. Specifically, EPC treatment was associated with the restoration of renal microcirculation and improved renal function. EPC therapy was most efficient when cells were delivered embedded in HA hydrogel. These findings establish major therapeutic benefits of adoptive transfer of EPCs, especially when embedded in HA hydrogels, in mice with LPS-induced endotoxemia, and they argue that hemodynamic and renal abnormalities of endotoxemia are in significant part due to developing incompetence of endogenous EPCs.
Assuntos
Células Endoteliais/transplante , Endotoxemia/terapia , Hidrogéis/administração & dosagem , Rim/irrigação sanguínea , Transplante de Células-Tronco , Animais , Pressão Sanguínea/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Microcirculação , Neovascularização Fisiológica , Inclusão do TecidoRESUMO
We developed an ex vivo approach characterizing renal mesenchymal stem cell (MSC) adhesion to kidney sections. Specificity of MSC adhesion was confirmed by demonstrating a) 3T3 cells displayed 10-fold lower adhesion, and b) MSC adhesion was CXCR4/stromal-derived factor-1 (SDF-1)-dependent. MSC adhesion was asymmetrical, with postischemic sections exhibiting more than twofold higher adhesion than controls, and showed preference to perivascular areas. Pretreating kidney sections with cyclic arginine-glycine-aspartic acid peptide resulted in increased MSC adhesion (by displacing resident cells), whereas blockade of CXCR4 with AMD3100 and inhibition of alpha4beta1(VLA4) integrin or vascular cellular adhesion molecule-1, reduced adhesion. The difference between adhered cells under cyclic arginine-glycine-aspartic acid peptide-treated and control conditions reflected prior occupancy of binding sites with endogenous cells. The AMD3100-inhibitable fraction of adhesion reflected CXCR4-dependent adhesion, whereas maximal adhesion was interpreted as kidney MSC-lodging capacity. MSC obtained from mice overexpressing caveolin-1 exhibited more robust adhesion than those obtained from knockout animals, consistent with CXCR4 dimerization in caveolae. These data demonstrate a) CXCR4/SDF-1-dependent adhesion increases in ischemia; b) CXCR4/SDF-1 activation is dependent on MSC surface caveolin-1; and c) occupancy of MSC binding sites is decreased, while d) capacity of MSC binding sites is expanded in postischemic kidneys. In conclusion, we developed a cell-bait strategy to unmask renal stem cell binding sites, which may potentially shed light on the MSC niche(s) and its characteristics.