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1.
Am J Physiol Renal Physiol ; 327(1): F103-F112, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38779750

RESUMO

α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.NEW & NOTEWORTHY A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.


Assuntos
alfa-Globulinas , Rim , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/tratamento farmacológico , alfa-Globulinas/metabolismo , alfa-Globulinas/farmacologia , Masculino , Rim/efeitos dos fármacos , Rim/patologia , Rim/metabolismo , Modelos Animais de Doenças , Taxa de Filtração Glomerular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Humanos , Camundongos , Heme Oxigenase-1/metabolismo , Ratos , Ratos Sprague-Dawley , Injúria Renal Aguda/patologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/prevenção & controle , Distribuição Tecidual
2.
Eur J Immunol ; 51(2): 354-367, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32926407

RESUMO

Peritoneal dialysis (PD) employs hypertonic glucose to remove excess water and uremic waste. Peritoneal membrane failure limits its long-term use. T-cell cytokines promote this decline. T-cell differentiation is critically determined by the microenvironment. We here study how PD-range hypertonic glucose regulates T-cell polarization and IL-17 production. In the human peritoneal cavity, CD3+ cell numbers increased in PD. Single cell RNA sequencing detected expression of T helper (Th) 17 signature genes RORC and IL23R. In vitro, PD-range glucose stimulated spontaneous and amplified cytokine-induced Th17 polarization. Osmotic controls l-glucose and d-mannose demonstrate that induction of IL-17A is a substance-independent, tonicity dose-dependent process. PD-range glucose upregulated glycolysis and increased the proportion of dysfunctional mitochondria. Blockade of reactive-oxygen species (ROS) prevented IL-17A induction in response to PD-range glucose. Peritoneal mesothelium cultured with IL-17A or IL17F produced pro-inflammatory cytokines IL-6, CCL2, and CX3CL1. In PD patients, peritoneal IL-17A positively correlated with CX3CL1 concentrations. PD-range glucose-stimulated, but neither identically treated Il17a-/- Il17f-/- nor T cells cultured with the ROS scavenger N-acetylcysteine enhanced mesothelial CX3CL1 expression. Our data delineate PD-range hypertonic glucose as a novel inducer of Th17 polarization in a mitochondrial-ROS-dependent manner. Modulation of tonicity-mediated effects of PD solutions may improve membrane survival.


Assuntos
Epitélio/imunologia , Glucose/imunologia , Inflamação/imunologia , Interleucina-17/imunologia , Peritônio/imunologia , Células Th17/imunologia , Animais , Células Cultivadas , Quimiocina CCL2/imunologia , Quimiocina CXCL1/imunologia , Feminino , Humanos , Interleucina-6/imunologia , Masculino , Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/imunologia , Diálise Peritoneal/métodos , Espécies Reativas de Oxigênio/imunologia , Regulação para Cima/imunologia
3.
FASEB J ; 34(12): 16307-16318, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33089923

RESUMO

Postischemic acute kidney injury (AKI) is a common clinical complication and often fatal, with no effective treatment available. Little is known about the role of leukocytes trapped in renal vessels during ischemia-reperfusion injury (IRI) in the postischemic AKI. We designed a new animal model in rats with preforming renal artery lavage prior to IRI to investigate the effect of diminishing the residual circulating leukocytes on kidney damage and inflammation. Moreover, the functional changes of macrophages in hypoxia reoxygenation condition were also analyzed. We found pre-ischemic renal lavage significantly decreased the serum creatinine and blood urea nitrogen levels, and downregulated the mRNA and protein expressions in kidneys and urinary secretion of kidney injury molecule-1 of rats after IRI. The renal pathological damage caused by IRI was also ameliorated by pre-ischemic renal lavage, as evidenced by fewer cast formation, diminished morphological signs of AKI in the tissue at 24 hours after IRI. Pre-ischemic renal lavage reduced the numbers of infiltrating CD68+ macrophages and MPO+ neutrophils. The mRNA expression of pro-inflammatory mediator in IRI kidneys and the levels of pro-inflammatory cytokines in circulatory system and urine were also reduced due to pre-ischemic lavage. Compared with nontreated rats with IRI, pre-ischemic renal lavage significantly reduced the phosphorylation levels of ERK and p65 subunit of NF-κB in the kidney after IRI. In addition, we found hypoxia/reoxygenation could promote the expression of pro-inflammatory mediators and inhibit the expression of anti-inflammatory factors by regulating ERK/NF-κB signaling pathway. Thus, pre-ischemic renal lavage could clearly reduce the renal damage after IRI by attenuating inflammation, and macrophages trapped in renal vessels during IRI could be important pathogenic factors driving tissue injury.


Assuntos
Injúria Renal Aguda/patologia , Inflamação/patologia , Rim/patologia , Traumatismo por Reperfusão/patologia , Injúria Renal Aguda/metabolismo , Animais , Nitrogênio da Ureia Sanguínea , Linhagem Celular , Creatinina/metabolismo , Inflamação/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Artéria Renal/metabolismo , Artéria Renal/patologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologia
4.
J Immunol ; 203(8): 2291-2300, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31527197

RESUMO

Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38MAPK/MAPKAPK2/3-mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow-derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa. The comparisons reveal that MK2/3 are the only LPS-induced kinases for S52 and S178 of TTP and the role of MK2 and MK3 in the regulation of TNF biosynthesis is not restricted to phosphorylation of TTP at S52/S178 but includes independent processes, which could involve other TTP phosphorylations (such as S316) or other substrates of MK2/3 or p38MAPK Furthermore, we found differences in the dependence of various cytokines on the cooperation between MK2/3 deletion and TTP mutation ex vivo. In the cecal ligation and puncture model of systemic inflammation, a dramatic decrease of cytokine production in MK2/3 DKO, TTPaa, and DKO/TTPaa mice compared with wild-type animals is observed, thus confirming the role of the MK2/3/TTP signaling axis in cytokine production also in vivo. These findings improve our understanding of this signaling axis and could be of future relevance in the treatment of inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/biossíntese , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência
5.
Kidney Int ; 95(6): 1405-1417, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30948201

RESUMO

Peritoneal dialysis (PD) is limited by chronic fibrotic remodeling of the peritoneal wall, a transforming growth factor-ß (TGF-ß)-mediated process. The fractalkine (CX3CL1) receptor CX3CR1 is expressed on macrophages and monocytes, where it is a marker of TGFß expression. Detection of its ligand CX3CL1 on the peritoneal mesothelium led us to hypothesize a pathophysiologic role of CX3CL1-CX3CR1 interaction in peritoneal fibrosis. We found that CX3CL1 was expressed on peritoneal mesothelial cells from PD patients and in a murine PD model. CX3CR1, mostly expressed on macrophages in the peritoneal wall, promoted fibrosis induced by chronic dialysate exposure in the mouse model. Our data suggest a positive feedback loop whereby direct interaction with CX3CR1-expressing macrophages promotes mesothelial expression of CX3CL1 and TGFß expression. In turn, TGFß upregulates CX3CR1 in murine and human monocytic cells. Upstream, macrophage cytokines including interleukin-1ß (IL-1ß) promote mesothelial CX3CR1 and TGFß expression, providing a starting point for CX3CL1-CX3CR1 interaction. IL-1ß expression was enhanced by exposure to dialysate both in vitro and in the mouse models. Our data suggest that macrophage-mesothelial cell crosstalk through CX3CR1-CX3CL1 interaction enhances mesothelial TGFß production, promoting peritoneal fibrosis in response to dialysate exposure. This interaction could be a novel therapeutic target in PD-associated chronic peritoneal fibrosis.


Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Fibrose Peritoneal/patologia , Idoso , Animais , Comunicação Celular , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Soluções para Diálise/toxicidade , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/etiologia , Peritônio/citologia , Peritônio/patologia , Cultura Primária de Células , Insuficiência Renal Crônica/terapia , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
6.
Nephrol Dial Transplant ; 34(6): 947-960, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30247663

RESUMO

BACKGROUND: Peritoneal membrane (PM) damage during peritoneal dialysis (PD) is mediated largely by high glucose (HG)-induced pro-inflammatory and neo-angiogenic processes, resulting in PM fibrosis and ultrafiltration failure. We recently demonstrated a crucial role for protein kinase C (PKC) isoform α in mesothelial cells. METHODS: In this study we investigate the role of PKCß in PM damage in vitro using primary mouse peritoneal macrophages (MPMΦ), human macrophages (HMΦ) and immortalized mouse peritoneal mesothelial cells (MPMCs), as well as in vivo using a chronic PD mouse model. RESULTS: We demonstrate that PKCß is the predominant classical PKC isoform expressed in primary MPMΦ and its expression is up-regulated in vitro under HG conditions. After in vitro lipopolysaccharides stimulation PKCß-/- MPMΦ demonstrates increased levels of interleukin 6 (IL-6), tumour necrosis factor α, and monocyte chemoattractant protein-1 and drastically decrease IL-10 release compared with wild-type (WT) cells. In vivo, catheter-delivered treatment with HG PD fluid for 5 weeks induces PKCß up-regulation in omentum of WT mice and results in inflammatory response and PM damage characterized by fibrosis and neo-angiogenesis. In comparison to WT mice, all pathological changes are strongly aggravated in PKCß-/- animals. Underlying molecular mechanisms involve a pro-inflammatory M1 polarization shift of MPMΦ and up-regulation of PKCα in MPMCs of PKCß-/- mice. Finally, we demonstrate PKCß involvement in HG-induced polarization processes in HMΦ. CONCLUSIONS: PKCß as the dominant PKC isoform in MPMΦ is up-regulated by HG PD fluid and exerts anti-inflammatory effects during PD through regulation of MPMΦ M1/M2 polarization and control of the dominant mesothelial PKC isoform α.


Assuntos
Macrófagos/metabolismo , Diálise Peritoneal/efeitos adversos , Proteína Quinase C beta/deficiência , Animais , Quimiocina CCL2/metabolismo , Soluções para Diálise/metabolismo , Modelos Animais de Doenças , Células Epiteliais , Epitélio , Feminino , Glucose/metabolismo , Humanos , Inflamação , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Transgênicos , Neovascularização Patológica , Omento/metabolismo , Fibrose Peritoneal/metabolismo , Peritônio/metabolismo , Isoformas de Proteínas , Proteína Quinase C-alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
7.
Kidney Int ; 92(3): 646-656, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28396121

RESUMO

Chronic renal allograft dysfunction (CAD) is a major limiting factor of long-term graft survival. It is characterized by interstitial fibrosis and tubular atrophy. The underlying pathomechanisms are incompletely understood. MicroRNAs are powerful regulators of gene expression and may have an impact on various diseases by direct mRNA decay or translational inhibition. A murine model of allogenic kidney transplantation was used resulting in CAD at 6 weeks after kidney transplantation. We identified fibrosis-associated miR-21a-5p by whole miRNAome expression analysis to be among the most highly upregulated miRNAs. In vitro in renal fibroblasts, miR-21a-5p was transcriptionally activated by interleukin 6-induced signal transducer and activator of transcription 3. Co-culture of LPS-activated macrophages with renal fibroblasts increased expression levels of miR-21a-5p and markers of fibrosis and inflammation. In addition, mature miR-21a-5p was secreted by macrophages in small vesicles, which were internalized by renal fibroblasts, thereby promoting profibrotic and proinflammatory effects. Notch2 receptor was identified as a potential target of miR-21a-5p and validated by luciferase gene reporter assays. Therapeutic silencing of miR-21a-5p in mice after allogenic kidney transplantation resulted in an amelioration of CAD, as indicated by a reduction in fibrosis development, inflammatory cell influx, tissue injury and BANFF lesion scoring. In a life-supporting model, miR-21a-5p antagonism had beneficial effects on kidney function. miR-21a-5p silencing may therefore be a viable therapeutic option in the treatment of patients following kidney transplantation to halt the development of CAD.


Assuntos
Aloenxertos/patologia , Rejeição de Enxerto/genética , Transplante de Rim/efeitos adversos , Rim/patologia , MicroRNAs/metabolismo , Receptor Notch2/genética , Animais , Biomarcadores/metabolismo , Doença Crônica , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Fibroblastos , Fibrose , Perfilação da Expressão Gênica , Sobrevivência de Enxerto/genética , Humanos , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Oligonucleotídeos/genética , Receptor Notch2/metabolismo , Transplante Homólogo/efeitos adversos , Regulação para Cima
8.
Kidney Int ; 89(6): 1253-67, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27142955

RESUMO

Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model of peritoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage and T cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid-induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-ß1-induced epithelial-to-mesenchymal transition and peritoneal dialysis fluid-induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients.


Assuntos
Carbazóis/uso terapêutico , Soluções para Diálise/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Glucose/efeitos adversos , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/prevenção & controle , Proteína Quinase C-alfa/antagonistas & inibidores , Animais , Linhagem Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peritônio/citologia , Peritônio/patologia , Cultura Primária de Células , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima
9.
Inflamm Res ; 65(2): 133-42, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26645911

RESUMO

OBJECTIVE: The present study aimed to comprehensively investigate the changes in oxylipins during murine sepsis induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). METHODS: Twenty-four hours after induction of sepsis in male C57BL/6 mice by LPS or CLP, plasma and liver, lung, kidney and heart tissues were sampled. Oxylipin levels in plasma and tissue were quantified by means of LC-MS. Moreover, clinical chemistry parameters were determined in plasma and interleukin levels (MCP-1 and IL-6) were determined in kidney and liver. RESULTS: Elevation of liver function plasma parameters at 24 h revealed that both models were successful in the induction of sepsis. LPS induced sepsis resulted in a dramatic increase of plasma PGE2 (2,100% change in comparison to control) and other cyclooxygenase metabolites, whereas this effect was less pronounced in CLP induced sepsis (97% increase of PGE2). Plasma epoxy-fatty acids (FAs) and hydroxy-FAs and most of the dihydroxy-FAs were elevated in both models of sepsis. Changes of tissue oxylipin concentrations were organ dependent. Only few changes were detected in the lung and liver tissue, epoxy-FAs were elevated in the kidney. In the heart tissue a trend towards lower levels of hydroxy-FAs and epoxy-FAs was observed. CONCLUSION: Both murine models of sepsis are characterized by changes of oxylipins formed in all branches of the arachidonic acid (AA) cascade. The more pronounced effects in the LPS model make this model more suitable for the investigation of the AA cascade and its pharmacological modulation in sepsis.


Assuntos
Oxilipinas/sangue , Sepse/sangue , Alprostadil/sangue , Alprostadil/metabolismo , Animais , Ceco/cirurgia , Quimiocina CCL2/genética , Dinoprostona/sangue , Dinoprostona/metabolismo , Interleucina-6/genética , Rim/metabolismo , Ligadura , Lipopolissacarídeos , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Oxilipinas/metabolismo , RNA Mensageiro/metabolismo , Sepse/metabolismo
10.
Am J Physiol Renal Physiol ; 307(6): F718-26, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25056349

RESUMO

Acute kidney injury (AKI) increases the risk of morbidity and mortality after major surgery and transplantation. We investigated the effect of PKC-ε deficiency on AKI and ischemic allograft damage after kidney transplantation. PKC-ε-deficient and wild type (WT) control mice were subjected to 35 min of renal pedicle clamping to induce AKI. PKC-ε deficiency was associated with a marked improvement in survival and an attenuated loss of kidney function. Furthermore, functional MRI experiments revealed better renal perfusion in PKC-ε-deficient mice than in WT mice one day after IRI. Acute tubular necrosis and neutrophil infiltration were markedly reduced in PKC-ε-deficient mice. To determine whether this resistance to ischemia-reperfusion injury resulted from changes in local renal cells or infiltrating leukocytes, we studied a life-supporting renal transplant model of ischemic graft injury. We transplanted kidneys from H(2b) PKC-ε-deficient mice (129/SV) and their corresponding WT littermates into major histocompatibility complex-incompatible H(2d) recipients (BALB/c) and induced ischemic graft injury by prolonged cold ischemia time. Recipients of WT allografts developed severe renal failure and died within 10 days of transplantation. Recipients of PKC-ε-deficient allografts had better renal function and survival; they had less generation of ROS and upregulation of proinflammatory proteins (i.e., ICAM-1, inducible nitric oxide synthase, and TNF-α) and showed less tubular epithelial cell apoptosis and inflammation in their allografts. These data suggest that local renal PKC-ε expression mediates proapoptotic and proinflammatory signaling and that an inhibitor of PKC-ε signaling could be used to prevent hypoxia-induced AKI.


Assuntos
Injúria Renal Aguda/enzimologia , Proteína Quinase C-épsilon/metabolismo , Traumatismo por Reperfusão/enzimologia , Aloenxertos/enzimologia , Animais , Apoptose , Sobrevivência de Enxerto , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Testes de Função Renal , Transplante de Rim , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
J Immunol ; 188(8): 4043-51, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22403440

RESUMO

Protein kinase C (PKC)-θ is involved in T cell activation via regulating the avidity of the ß(2) integrin LFA-1 in the immunological synapse. LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-θ in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP(+) (wild-type [WT]) and PKC-θ gene-deficient (Prkcq(-/-)) mice. Following stimulation with CXCL1, both WT and Prkcq(-/-) cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq(-/-) neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq(-/-) neutrophils started 30 s after CXCL1 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq(-/-) neutrophils was delayed. Prkcq(-/-) neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced peritonitis, and LPS-induced lung injury. We conclude that PKC-θ mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-θ in outside-in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo.


Assuntos
Antígenos CD18/imunologia , Isoenzimas/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Neutrófilos/imunologia , Proteína Quinase C/imunologia , Vênulas/imunologia , Animais , Antígenos CD18/genética , Adesão Celular , Quimiocina CXCL1/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Sinapses Imunológicas , Isoenzimas/deficiência , Isoenzimas/genética , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Vídeo , Neutrófilos/citologia , Neutrófilos/fisiologia , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C-theta , Vênulas/citologia , Vênulas/metabolismo
12.
Mol Ther ; 21(12): 2236-46, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23887360

RESUMO

Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Complemento C5a/antagonistas & inibidores , Insuficiência de Múltiplos Órgãos/prevenção & controle , Peritonite/tratamento farmacológico , Sepse/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Aptâmeros de Nucleotídeos/farmacocinética , Complemento C5a/imunologia , Complemento C5a/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peritonite/complicações , Peritonite/fisiopatologia , Sepse/complicações
13.
J Leukoc Biol ; 115(3): 573-582, 2024 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-38038378

RESUMO

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.


Assuntos
Monócitos , Receptor de Fator Estimulador de Colônias de Macrófagos , Camundongos , Animais , Monócitos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Citometria de Fluxo , Macrófagos/metabolismo , Diferenciação Celular
14.
Front Immunol ; 15: 1368040, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38562925

RESUMO

Background: Excessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis. Methods: Polymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC). Results: All AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro. Conclusion: Data from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.


Assuntos
Doenças Transmissíveis , Sepse , Humanos , Camundongos , Animais , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Quimiocinas , Fatores Imunológicos
15.
Clin Transl Sci ; 16(12): 2729-2743, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37899696

RESUMO

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.


Assuntos
Injúria Renal Aguda , Hemopexina , Traumatismo por Reperfusão , Animais , Humanos , Camundongos , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Ponte Cardiopulmonar/efeitos adversos , Heme , Hemoglobinas/metabolismo , Hemólise , Hemopexina/química , Hemopexina/metabolismo , Isquemia/complicações , Rim/metabolismo , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/etiologia
16.
J Am Soc Nephrol ; 22(10): 1887-96, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21841063

RESUMO

Renal ischemia-reperfusion contributes to reduced renal allograft survival. The peptide Bß(15-42), a breakdown product of fibrin, attenuates inflammation induced by ischemia-reperfusion in the heart by competitively blocking the binding of leukocytes to endothelial VE-cadherin, but whether it could improve outcomes in renal transplantation is unknown. Here, we tested the ability of Bß(15-42) to ameliorate the effects of renal ischemic injury during allogenic kidney transplantation in mice. In our renal transplantation model (C57BL/6 into BALB/c mice), treatment with Bß(15-42) at the time of allograft reperfusion resulted in significantly improved survival of recipients during the 28-day follow-up (60% versus 10%). Bß(15-42) treatment decreased leukocyte infiltration, expression of endothelial adhesion molecules, and proinflammatory cytokines. Treatment significantly attenuated allogenic T cell activation and reduced cellular rejection. Moreover, Bß(15-42) significantly reduced tubular epithelial damage and apoptosis, which we reproduced in vitro. These data suggest that Bß(15-42) may have therapeutic potential in transplant surgery by protecting grafts from ischemia-reperfusion injury.


Assuntos
Injúria Renal Aguda/prevenção & controle , Produtos de Degradação da Fibrina e do Fibrinogênio/administração & dosagem , Transplante de Rim/efeitos adversos , Fragmentos de Peptídeos/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Feminino , Transplante de Rim/imunologia , Túbulos Renais/citologia , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Transplante Homólogo
17.
Sci Rep ; 12(1): 16878, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207386

RESUMO

Recent reports demonstrate that SARS-CoV-2 utilizes cell surface heparan sulfate as an attachment factor to facilitate the initial interaction with host cells. Heparan sulfate interacts with the receptor binding domain of SARS-CoV-2 spike glycoprotein, and blocking this interaction can decrease cell infection. We and others reported recently that the family of compounds of 2,5-dihydroxyphenylic acid interferes with the binding of the positively charged groove in growth factor molecules to negatively charged cell surface heparan sulfate. We hypothesized that Calcium Dobesilate (CaD)-calcium salt of 2,5-dihydroxyphenylic acid-may also interfere with the binding of SARS-CoV-2 spike protein to heparan sulfate. Using lentiviral SARS-CoV-2 spike protein pseudotyped particles we show that CaD could significantly reduce pseudovirus uptake into endothelial cells. On the contrary, CaD did not affect cell infection with VSVG-expressing lentivirus. CaD could also prevent retention of SARS-CoV-2 spike protein in ex vivo perfused mouse kidney. Using microfluidic culture of endothelial cells under flow, we show that CaD prevents spike protein interaction with heparan sulfate glycocalyx. Since CaD has no adverse side effects and is approved in humans for other medical indications, our findings can rapidly translate into clinical studies.


Assuntos
Tratamento Farmacológico da COVID-19 , Dobesilato de Cálcio , Animais , Cálcio/metabolismo , Células Endoteliais/metabolismo , Heparitina Sulfato/metabolismo , Heparitina Sulfato/farmacologia , Humanos , Camundongos , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral
18.
Biomedicines ; 10(5)2022 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-35625934

RESUMO

Nephrotoxic drugs can cause acute kidney injury (AKI) and analgesic nephropathy. Diclofenac is potentially nephrotoxic and frequently prescribed for pain control. In this study, we investigated the effects of single and repetitive oral doses of diclofenac in the setting of pre-existing subclinical AKI on the further course of AKI and on long-term renal consequences. Unilateral renal ischemia-reperfusion injury (IRI) for 15 min was performed in male CD1 mice to induce subclinical AKI. Immediately after surgery, single oral doses (100 mg or 200 mg) of diclofenac were administered. In a separate experimental series, repetitive treatment with 100 mg diclofenac over three days was performed after IRI and sham surgery. Renal morphology and pro-fibrotic markers were investigated 24 h and two weeks after the single dose and three days after the repetitive dose of diclofenac treatment using histology, immunofluorescence, and qPCR. Renal function was studied in a bilateral renal IRI model. A single oral dose of 200 mg, but not 100 mg, of diclofenac after IRI aggravated acute tubular injury after 24 h and caused interstitial fibrosis and tubular atrophy two weeks later. Repetitive treatment with 100 mg diclofenac over three days aggravated renal injury and caused upregulation of the pro-fibrotic marker fibronectin in the setting of subclinical AKI, but not in sham control kidneys. In conclusion, diclofenac aggravated renal injury in pre-existing subclinical AKI in a dose and time-dependent manner and already a single dose can cause progression to chronic kidney disease (CKD) in this model.

19.
J Vis Exp ; (185)2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35938826

RESUMO

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.


Assuntos
Transplante de Rim , Ureter , Anastomose Cirúrgica/métodos , Animais , Transplante de Rim/métodos , Camundongos , Artéria Renal/cirurgia , Ureter/cirurgia , Bexiga Urinária/cirurgia
20.
Front Immunol ; 13: 846695, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432371

RESUMO

Background: Acute rejection (AR) in kidney transplantation is an established risk factor that reduces the survival rate of allografts. Despite standard immunosuppression, molecules with regulatory control in the immune pathway of AR can be used as important targets for therapeutic operations to prevent rejection. Methods: We downloaded the microarray data of 15 AR patients and 37 non-acute rejection (NAR) patients from Gene Expression Omnibus (GEO). Gene network was constructed, and genes were classified into different modules using weighted gene co-expression network analysis (WGCNA). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cytoscape were applied for the hub genes in the most related module to AR. Different cell types were explored by xCell online database and single-cell RNA sequencing. We also validated the SLAMF8 and TLR4 levels in Raw264.7 and human kidney tissues of TCMR. Results: A total of 1,561 differentially expressed genes were filtered. WGCNA was constructed, and genes were classified into 12 modules. Among them, the green module was most closely associated with AR. These genes were significantly enriched in 20 pathway terms, such as cytokine-cytokine receptor interaction, chemokine signaling pathway, and other important regulatory processes. Intersection with GS > 0.4, MM > 0.9, the top 10 MCC values and DEGs in the green module, and six hub genes (DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE) were identified. Their expression levels were all confirmed to be significantly elevated in AR patients in GEO, Nephroseq, and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing showed that AR patient had a higher percentage of native T, CD1C+_B DC, NKT, NK, and monocytes in peripheral blood mononuclear cells (PBMCs). Xcell enrichment scores of 20 cell types were significantly different (p<0.01), mostly immune cells, such as B cells, CD4+ Tem, CD8+ T cells, CD8+ Tcm, macrophages, M1, and monocytes. GSEA suggests that highly expressed six hub genes are correlated with allograft rejection, interferon γ response, interferon α response, and inflammatory response. In addition, SLAMF8 is highly expressed in human kidney tissues of TCMR and in M1 phenotype macrophages of Raw264.7 cell line WGCNA accompanied by high expression of TLR4. Conclusion: This study demonstrates six hub genes and functionally enriched pathways related to AR. SLAMF8 is involved in the M1 macrophages via TLR4, which contributed to AR process.


Assuntos
Transplante de Rim , Receptor 4 Toll-Like , Redes Reguladoras de Genes , Humanos , Transplante de Rim/efeitos adversos , Leucócitos Mononucleares , Macrófagos , Proteínas de Membrana , Família de Moléculas de Sinalização da Ativação Linfocitária/genética
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