RESUMO
The biomedical consequences of allogeneic blood transfusions and the possible pathomechanisms of transfusion-related morbidity and mortality are still not entirely understood. In retrospective studies, allogeneic transfusion was associated with increased rates of cancer recurrence, metastasis and death in patients with colorectal cancer. However, correlation does not imply causation. The purpose of this study was to elucidate this empirical observation further in order to address insecurity among patients and clinicians. We focused on the in vitro effect of microparticles derived from red blood cell units (RMPs). We incubated different colon carcinoma cells with RMPs and analyzed their effects on growth, invasion, migration and tumor marker expression. Furthermore, effects on Wnt, Akt and ERK signaling were explored. Our results show RMPs do not seem to affect functional and phenotypic characteristics of different colon carcinoma cells and did not induce or inhibit Wnt, Akt or ERK signaling, albeit in cell culture models lacking tumor microenvironment. Allogeneic blood transfusions are associated with poor prognosis, but RMPs do not seem to convey tumor-enhancing effects. Most likely, the circumstances that necessitate the transfusion, such as preoperative anemia, tumor stage, perioperative blood loss and extension of surgery, take center stage.
Assuntos
Carcinoma , Micropartículas Derivadas de Células , Neoplasias do Colo , Carcinoma/complicações , Micropartículas Derivadas de Células/patologia , Neoplasias do Colo/patologia , Humanos , Recidiva Local de Neoplasia/etiologia , Proteínas Proto-Oncogênicas c-akt , Estudos Retrospectivos , Microambiente TumoralRESUMO
BACKGROUND: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. METHODS: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. RESULTS: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. CONCLUSIONS: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.
Assuntos
Carcinoma de Células Renais/metabolismo , Ferro/metabolismo , Neoplasias Renais/metabolismo , Lipocalina-2/metabolismo , RNA Mensageiro/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Humanos , Técnicas In Vitro , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Lipocalina-2/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria Atômica , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Lipocalin-2 (Lcn-2) is rapidly upregulated in macrophages after renal tubular injury and acts as renoprotective and pro-regenerative agent. Lcn-2 possesses the ability to bind and transport iron with high affinity. Therefore, the present study focuses on the decisive role of the Lcn-2 iron-load for its pro-regenerative function. Primary mouse tubular epithelial cells were isolated from kidney tissue of wildtype mice and incubated with 5µM Cisplatin for 24h to induce injury. Bone marrow-derived macrophages of wildtype and Lcn-2-/- mice were isolated and polarized with IL-10 towards an anti-inflammatory, iron-release phenotype. Their supernatants as well as recombinant iron-loaded holo-Lcn-2 was used for stimulation of Cisplatin-injured tubular epithelial cells. Incubation of tubular epithelial cells with wildtype supernatants resulted in less damage and induced cellular proliferation, whereas in absence of Lcn-2 no protective effect was observed. Epithelial integrity as well as cellular proliferation showed a clear protection upon rescue experiments applying holo-Lcn-2. Notably, we detected a positive correlation between total iron amounts in tubular epithelial cells and cellular proliferation, which, in turn, reinforced the assumed link between availability of Lcn-2-bound iron and recovery. We hypothesize that macrophage-released Lcn-2-bound iron is provided to tubular epithelial cells during toxic cell damage, whereby injury is limited and recovery is favored.
Assuntos
Células Epiteliais/metabolismo , Rim/metabolismo , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Regeneração , Animais , Proliferação de Células , Cisplatino/efeitos adversos , Ferro/metabolismo , Rim/efeitos dos fármacos , Rim/lesões , Lipocalina-2/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes , Regulação para CimaRESUMO
It is well known that sepsis and inflammation reduce male fertility. Within the testis, toll-like receptor 3 (TLR3) is constitutively expressed and recognizes double-stranded RNA (dsRNA) from viruses, degraded bacteria, damaged tissues and necrotic cells. To characterize the potential role of TLR3 in response to testicular infections, its expression and downstream signaling were investigated upon challenge with lipopolysaccharides (LPS) in two mouse strains that differ in their immuno-competence regarding T cell-regulated immunity. Thereto, Balb/c and Foxn1nu mice were randomized into six interventional groups treated with either i.v. application of saline or LPS followed by 20 min, 5 h 30 min and 18 h of observation and two sham-treated control groups. LPS administration induced a significant stress response; the amplification was manifested for TLR3 and interleukin 6 (IL6) mRNA in the impaired testis 5 h 30 min after LPS injection. TLR3 immunostaining revealed that TLR3 was primarily localized in spermatocytes. The TLR3 expression displayed different temporal dynamics between both mouse strains. However, immunofluorescence staining indicated only punctual interferon regulatory factor 3 (IRF3) expression upon LPS treatment along with minor alterations in interferon ß (IFNß) mRNA expression. Induction of acute inflammation was closely followed by a significant shift of the Bax/Bcl2 ratio to pro-apoptotic signaling accompanied by augmented TUNEL-positive cells 18 h after LPS injection with again differing patterns in both mouse strains. In conclusion, this study shows the involvement of TLR3 in response to LPS-induced testicular inflammation in immuno-competent and -incompetent mice, yet lacking transmission into its signaling pathway.
Assuntos
Apoptose/imunologia , Orquite/imunologia , Espermatócitos/imunologia , Testículo/metabolismo , Receptor 3 Toll-Like/imunologia , Animais , Fator Regulador 3 de Interferon/imunologia , Interferon beta/imunologia , Lipopolissacarídeos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Orquite/induzido quimicamente , Espermatócitos/citologia , Testículo/patologiaRESUMO
Stem cell-based therapies require cells with a maximum regenerative capacity in order to support regeneration after tissue injury and organ failure. Optimization of this regenerative potential of mesenchymal stromal/stem cells (MSC) or their conditioned medium by in vitro preconditioning regimens are considered to be a promising strategy to improve the release of regenerative factors. In the present study, MSC were isolated from inguinal adipose tissue (mASC) from C57BL/6 mice, cultured, and characterized. Then, mASC were either preconditioned by incubation in a hypoxic environment (0.5% O2), or in normoxia in the presence of murine epidermal growth factor (EGF) or tumor necrosis factor α (TNFα) for 48 h. Protein expression was measured by a commercially available array. Selected factors were verified by PCR analysis. The expression of 83 out of 308 proteins (26.9%) assayed was found to be increased after preconditioning with TNFα, whereas the expression of 61 (19.8%) and 70 (22.7%) proteins was increased after incubation with EGF or in hypoxia, respectively. Furthermore, we showed the proliferation-promoting effects of the preconditioned culture supernatants on injured epithelial cells in vitro. Our findings indicate that each preconditioning regimen tested induced an individual expression profile with a wide variety of factors, including several growth factors and cytokines, and therefore may enhance the regenerative potential of mASC for cell-based therapies.
Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The pathomechanisms of morbidity due to blood transfusions are not yet entirely understood. Elevated levels of red blood cell-derived microparticles (RMPs) are found in coagulation-related pathologies and also in stored blood. Previous research has shown that RMPs mediate transfusion-related complications by the intrinsic pathway. We hypothesized that RMPs might play a role in post-transfusion thrombotic complications by enhancing procoagulant activity also through the extrinsic pathway of coagulation. STUDY DESIGN AND METHODS: In this laboratory study, blood from 18 healthy volunteers was stimulated with microparticles from expired stored red blood cells. Various clotting parameters were recorded. Flow cytometry, enzyme-linked immunosorbent assays, and real-time polymerase chain reaction were used to investigate possible mediating mechanisms. RESULTS: The addition of RMPs shortened the clotting time from 194 to 161 seconds (p < 0.001). After incubation with RMPs, there was increased expression of tissue factor (TF) on monocytes and in plasma. TF messenger RNA expression increased in a time-dependent and concentration-dependent manner. There was a significant induction of interleukin-1ß and interleukin-6. After stimulation with RMPs, there was a significant increase in the number of activated platelets, an increased percentage of PAC-1/CD62P (procaspase activating compound-1/platelet surface P-selectin) double-positive platelets, and an increased number of platelet-neutrophil duplets and platelet-monocyte duplets, indicating enhanced interaction of platelets with neutrophils and monocytes. Levels of CXCL-8 (C-X-C motif chemokine ligand 1) and interleukin-6 were significantly higher after treatment with RMPs. CONCLUSION: Our results suggest that RMPs trigger coagulation through TF signaling, induce the secretion of proinflammatory cytokines, and induce cell-cell interaction between platelets and neutrophils. Thus, under certain conditions, RMPs could play a role in post-transfusion complications through these mechanisms.
Assuntos
Coagulação Sanguínea , Micropartículas Derivadas de Células/fisiologia , Eritrócitos/citologia , Inflamação , Reação Transfusional , Preservação de Sangue , Comunicação Celular , Eritrócitos/ultraestrutura , Humanos , Inflamação/etiologia , Interleucina-6/sangue , Interleucina-8/sangue , Monócitos/metabolismo , Ativação Plaquetária , RNA Mensageiro/sangue , Tromboplastina/genética , Tromboplastina/metabolismo , Trombose/etiologiaRESUMO
The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is a cytokine-activated transcription factor controlling inflammation, cell proliferation, survival, and differentiation in normal tissue as well as in tumor growth. One of its most important negative regulators is the suppressor of cytokine signaling 3 (SOCS3). Here, we analyzed SOCS3 and other tumor-associated local immune regulators in human clear cell renal cell carcinoma (ccRCC). Analyses were performed in tumor and adjacent tumor-free healthy renal tissue from 35 patients with ccRCC. For functional analysis, ccRCC Caki-1 cell lines were stimulated with IL-6 and IFNγ in cell culture assays. We observed significantly lower SOCS3 messenger RNA (mRNA) levels in tumor tissue compared to healthy tissue. SOCS3 mRNA strongly correlated within tumor and healthy tissue. Interestingly vice versa, SOCS3 protein levels were significantly higher in tumor tissue than in healthy tissue. IL-22 and IL-22R1 mRNA displayed no differences in tumor and healthy tissue. Stimulation of Caki-1 cells with IFNγ resulted in markedly increased SOCS3 mRNA levels. We conclude that SOCS3 along with STAT3 participates in regulatory mechanisms in ccRCC, which certainly features only one of multiple factors involved but nevertheless merits further attention.
Assuntos
Carcinoma de Células Renais/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/genética , Humanos , Inflamação/genética , Interferon gama/genética , Interleucina-6/genética , Interleucinas/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Fator de Transcrição STAT3/genética , Interleucina 22RESUMO
Expression of kidney injury molecule-1 (Kim-1) is rapidly upregulated following tubular injury, constituting a biomarker for acute kidney damage. We examined the renal localization of Kim-1 expression in PKD/Mhm (polycystic kidney disease, Mannheim) (cy/+) rats (cy: mutated allel, +: wild type allel), an established model for autosomal dominant polycystic kidney disease, with chronic, mainly proximal tubulointerstitial alterations. For immunohistochemistry or Western blot analysis, kidneys of male adult heterozygously-affected (cy/+) and unaffected (+/+) littermates were perfusion-fixed or directly removed. Kim-1 expression was determined using peroxidase- or fluorescence-linked immunohistochemistry (alone or in combination with markers for tubule segments or differentiation). Compared to (+/+), only in (cy/+) kidneys, a chronic expression of Kim-1 could be detected by Western blot analysis, which was histologically confined to an apical cellular localization in areas of cystically-transformed proximal tubules with varying size and morphology, but not in distal tubular segments. Kim-1 was expressed by cystic epithelia exhibiting varying extents of dedifferentiation, as shown by double labeling with aquaporin-1, vimentin or osteopontin, yielding partial cellular coexpression. In this model, in contrast to other known molecules indicating renal injury and/or repair mechanisms, the chronic renal expression of Kim-1 is strictly confined to proximal cysts. Its exact role in interfering with tubulo-interstitial alterations in polycystic kidney disease warrants future investigations.
Assuntos
Moléculas de Adesão Celular/metabolismo , Túbulos Renais Proximais/metabolismo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Animais , Biomarcadores/metabolismo , Desdiferenciação Celular , Modelos Animais de Doenças , Túbulos Renais Proximais/patologia , Masculino , Especificidade de Órgãos , Ratos , Regulação para CimaRESUMO
The disruption of endothelial integrity is a crucial step for the development of vascular leakage and consequently ischemia-reperfusion injury (IRI). Regarding the molecular cell-cell interaction, the fibrinopeptide Bß15-42 prevents vascular leakage by stabilizing the inter-endothelial junctions via association with the vascular endothelial-cadherin. In a previous study we showed that a renoprotective effect in early IRI may be achieved by intravenous administration of Bß15-42 at the time of reperfusion. We now aimed to investigate whether additional pre-ischemic application of Bß15-42 could enhance this effect. Therefore C57BL/6 mice were subjected to 0.5h bilateral renal ischemia followed by reperfusion. The animals were randomized into 6 groups (n=6): two control groups treated with i.v. administration of NaCl at reperfusion for 0.5h (NaCl 1h) and 2.5h (NaCl 3h), two groups with Bß15-42 at reperfusion for 0.5h (Bß(rep) 1h) and 2.5h (Bß(rep) 3h), and two groups with administration of Bß15-42 immediately pre-ischemic as well as at reperfusion for 0.5h (Bß(peri) 1h) and 2.5h (Bß(peri) 3h). We found that both Bß(rep) and Bß(peri) mice displayed reduced early renal damage compared with NaCl treated mice. However, there was no further reduction of the IR damage through added pre-ischemic application of Bß15-42. Overall, we detected significantly reduced endothelial activation, lower tissue infiltration of neutrophils as well as lower tissue levels of neutrophil gelatinase-associated lipocalin (NGAL) in all mice treated with Bß15-42 compared to mice treated with NaCl. Our data confirm the renoprotective effect of Bß15-42 in the early therapeutic treatment of acute kidney injury due to ischemia and reperfusion. However, a combined pre-and post-ischemic administration of Bß15-42 appears to provide no additional benefit compared with a sole administration at reperfusion.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/administração & dosagem , Isquemia/tratamento farmacológico , Rim/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Proteínas de Fase Aguda/metabolismo , Animais , Permeabilidade Capilar , Adesão Celular , Células Endoteliais/metabolismo , Imuno-Histoquímica , Inflamação/patologia , Rim/patologia , Lipocalina-2 , Lipocalinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/patologia , Proteínas Oncogênicas/metabolismo , Fatores de TempoRESUMO
BACKGROUND: We examined the value of the novel acute kidney injury (AKI) markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in acute postrenal impairment. These biomarkers have been evaluated in prerenal and intrarenal AKI so far, but not in human acute postrenal kidney injury. With regard to multimorbid and critically ill patients the discrimination of different AKI origins often remains a challenge. As the trend goes towards a diagnostic panel of AKI markers, we hereby aim to contribute to evaluate further options of discrimination in an observational case-control study. MATERIALS AND METHODS: Blood and urine samples were obtained from 53 patients with acute obstructive nephropathy secondary to ureteral calculi and 52 age-matched healthy controls. Serum NGAL (sNGAL), urinary NGAL (uNGAL) and urinary KIM-1 (uKIM-1) levels were determined using a commercially available ELISA kit, creatinine applying the Jaffé's method. RESULTS: While urinary levels of KIM-1 were not significantly different between patients with obstructive nephropathy and controls, a striking increase in sNGAL (P < 0·001) and uNGAL (P < 0·01) levels was detected in the obstructive nephropathy group. Within the obstructive nephropathy group, sNGAL (P = 0·01) and uNGAL (P = 0·049) but not uKIM-1 correlated positively with the white blood cell count and uNGAL correlated positively (P = 0·002) with the extent of leucocyturia. CONCLUSIONS: High levels of sNGAL and uNGAL observed in stone-induced acute obstructive nephropathy may represent a valuable marker of postrenal AKI. Low uKIM-1 levels may help to discriminate postrenal AKI events using a panel of markers in this setting.
Assuntos
Injúria Renal Aguda/diagnóstico , Proteínas de Fase Aguda/metabolismo , Lipocalinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Virais/metabolismo , Injúria Renal Aguda/etiologia , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Lipocalina-2 , Masculino , Cólica Renal/etiologia , Cálculos Ureterais/complicações , Obstrução Ureteral/etiologiaRESUMO
BACKGROUND: There is increasing evidence that vitamin D metabolites influence carcinogenesis. Besides its role in mineral homoeostasis, calcitriol, the active metabolite of vitamin D (1,25(OH)2 D3 ), is known to possess antiproliferative, proapoptotic and immunomodulatory effects in cancer. Concerning the synthesis of vitamin D, the hydroxylases CYP2R1, CYP27B1 and CYP24A1 play a critical role, and the latter molecule determines the biological half-life of 1,25(OH)2 D3 , which is synthesized in the proximal renal tubules. MATERIALS AND METHODS: The adjacency of these two biological processes prompted us to investigate the gene expression of CYP2R1, CYP27B1 and CYP24A1 in patients with ccRCC. Using RT-PCR, we retrospectively compared mRNA expression profiles from human ccRCC tumour samples with those derived from the corresponding adjacent healthy tissues (n = 30). RESULTS: We observed that all three genes (CYP2R1, CYP27B1 and CYP24A1) were upregulated in tumours compared with normal tissue (P < 0·0001). Moreover, CYP24A1 displayed a significantly higher expression in tumours than CYP27B1 (P < 0·05) and CYP2R1 (P < 0·0001), whereas no differences in the expression of these genes were found in healthy renal tissue. Gene expression of CYP2R1, CYP27B1 and CYP24A did not differ between pathological classifications (TNM, grading, presence of metastasis). CONCLUSION: We thus conclude that upregulated gene expression of the catabolizing CYP24A1 as well as the synthesizing CYP2R1 and CYP27B1 may lead to a misbalance of vitamin D metabolites in ccRCC and thus contributing to its pathogenesis.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Carcinoma de Células Renais/genética , Colestanotriol 26-Mono-Oxigenase/genética , Neoplasias Renais/genética , Esteroide Hidroxilases/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Colestanotriol 26-Mono-Oxigenase/metabolismo , Família 2 do Citocromo P450 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Esteroide Hidroxilases/metabolismo , Regulação para Cima , Vitamina D3 24-HidroxilaseRESUMO
CONTEXT: Acute kidney injury (AKI) represents a common serious clinical problem. Up to date mortality due to AKI, especially in intensive care units, has not been changed significantly over the past 50 years. This is partly due to a delay in initiating renal protective and appropriate therapeutic measures since until now there are no reliable early-detecting biomarkers. The gold standard, serum creatinine, displays poor specificity and sensitivity with regard to recognition of the early period of AKI. OBJECTIVE: Our objective was to review established markers versus novel urine and serum biomarkers of AKI in humans, which have progressed to clinical phase with regard to their diagnostic and prognostic value. MATERIALS AND METHODS: A review was performed on the basis of literature search of renal failure, acute kidney injury, and biomarkers in Pubmed. RESULTS: Next to established biomarkers as creatinine and cystatin C, other molecules such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemotactic peptide (MCP-1), Netrin-1, and interleukin (IL)-18 are available and represent promising new markers that, however, need to be further evaluated in the clinical setting for suitability. DISCUSSION: In clinical settings with incipient AKI, not only the development and the implementation of more sensitive biomarkers are required for earlier treatment initiation in order to attenuate the severity of kidney injury, but also equally important remains the substantial improvement and application of refined and prophylactic therapeutic options in these situations. CONCLUSION: Adequately powered clinical trials testing a row of biomarkers are warranted before they may qualify for full adoption in clinical practice.
Assuntos
Injúria Renal Aguda/diagnóstico , Biomarcadores/sangue , Acetilglucosaminidase/urina , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Proteínas de Fase Aguda/urina , Animais , Nitrogênio da Ureia Sanguínea , Quimiocina CCL2/urina , Creatinina/sangue , Cistatina C/urina , Taxa de Filtração Glomerular , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Interleucina-18/urina , Lipocalina-2 , Lipocalinas/urina , Glicoproteínas de Membrana/urina , Fatores de Crescimento Neural/urina , Netrina-1 , Proteinúria/urina , Proteínas Proto-Oncogênicas/urina , Receptores Virais , Sensibilidade e Especificidade , Choque Séptico/complicações , Proteínas Supressoras de Tumor/urinaRESUMO
Accumulating evidence suggests that iron homeostasis is disturbed in tumors. We aimed at clarifying the distribution of iron in renal cell carcinoma (RCC). Considering the pivotal role of macrophages for iron homeostasis and their association with poor clinical outcome, we investigated the role of macrophage-secreted iron for tumor progression by applying a novel chelation approach. We applied flow cytometry and multiplex-immunohistochemistry to detect iron-dependent markers and analyzed iron distribution with atomic absorption spectrometry in patients diagnosed with RCC. We further analyzed the functional significance of iron by applying a novel extracellular chelator using RCC cell lines as well as patient-derived primary cells. The expression of iron-regulated genes was significantly elevated in tumors compared to adjacent healthy tissue. Iron retention was detected in tumor cells, whereas tumor-associated macrophages showed an iron-release phenotype accompanied by enhanced expression of ferroportin. We found increased iron amounts in extracellular fluids, which in turn stimulated tumor cell proliferation and migration. In vitro, macrophage-derived iron showed pro-tumor functions, whereas application of an extracellular chelator blocked these effects. Our study provides new insights in iron distribution and iron-handling in RCC. Chelators that specifically scavenge iron in the extracellular space confirmed the importance of macrophage-secreted iron in promoting tumor growth.
RESUMO
PURPOSE: NOD1 and NOD2 (nucleotide-binding oligomerization domain)-receptors are intracellular receptors and belong to the family of pattern recognition receptors being present in both human and murine renal tubular cells. Besides, NOD1 has been proved to promote apoptosis, upon its overexpression. Hence, we aimed to investigate NOD1 and NOD2 expression in human clear cell renal cell carcinoma (ccRCC). METHODS: Tumor and corresponding adjacent healthy tissues from 41 patients with histopathological diagnosis of ccRCC as well as primary isolated renal tubular epithelial cells (TECs) and tumor tissue from a murine xenograft model using CAKI-1 ccRCC cells were analyzed. RESULTS: NOD1 and NOD2 mRNA was constitutively expressed in both tumor and adjacent healthy renal tissue, with NOD1 being significantly lower and in contrast NOD2 significantly higher expressed in tumor tissue compared to healthy tissues. Immunohistochemically, NOD1 was located not only in the cytoplasm, but also in the nucleus in ccRCC tissue whereas NOD2 was solely localized in the cytoplasm in both human ccRCC as well as in the healthy tubular system. Focusing on the vasculature, NOD2 displayed broader expression than NOD1. In primary TECs as well as CAKI-1 cells NOD1 and NOD2 was constitutively expressed and increasable upon LPS stimulation. In the mouse xenograft model, human NOD1 mRNA was significantly higher expressed compared to NOD2. In contrast hereto, we observed a shift towards lower mouse NOD1 compared to NOD2 mRNA expression. CONCLUSION: In view of reduced apoptosis-associated NOD1 expression in ccRCC tissue opposed to higher expression of NOD2 in tumor vasculature, inducibility of NOD expression in TECs as well as the detected shift of NOD1 and NOD2 expression in the mouse xenograft model, modulation of NOD receptors might, therefore, provide a molecular therapeutic approach in ccRCC.
Assuntos
Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Xenoenxertos , Humanos , Imunidade Inata , Imuno-Histoquímica , Rim/irrigação sanguínea , Rim/imunologia , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/genética , Neoplasias Renais/patologia , Túbulos Renais/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD1/biossíntese , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/biossíntese , Proteína Adaptadora de Sinalização NOD2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The purpose of our study was to examine the nature and incidence of renal injuries during organ procurement, to identify risk factors and to analyse the effects of organ lesions on the following transplantation. METHODS: All cadaveric kidney transplantations performed at our centre from 1996 to 2006 with an organ donated within the Eurotransplant (ET) region were retrospectively analysed. RESULTS: Five hundred and sixty-three renal grafts procured in 62 centres throughout the ET region were transplanted in the analysed period. One hundred and twenty (21.3%) kidneys were inadequately procured with 143 errors in total. The frequency of procurement errors did not differ significantly between kidneys procured by urologists and general surgeons (19.2% vs. 24.6%) nor when kidneys were procured alone or together with pancreas and/or liver (19.3% vs. 22.0%). Inadequate procurement lead to a discard rate of 0.2% and ultimately resulted in a surgical complication rate of 3.4%. Primary graft function (75.8% vs. 78.6%), three-yr graft survival (76.6% vs. 82.4%) and cumulated long-term graft survival were not significantly influenced by procurement errors. CONCLUSION: Additional measures to improve procurement quality are necessary. Nevertheless, adequate repair of organ lesions is possible and most organs can be successfully transplanted with very good short- and long-term results.
Assuntos
Sobrevivência de Enxerto , Transplante de Rim , Rim/lesões , Obtenção de Tecidos e Órgãos , Adulto , Idoso , Idoso de 80 Anos ou mais , Cadáver , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida , Doadores de Tecidos , Resultado do TratamentoRESUMO
Shock wave therapy (SWT) has been shown to induce angiogenesis in ischaemic muscle. However, the mechanism of action remains unknown. Macrophages are crucial for angiogenic responses after ischaemic injury. The M2 macrophage subset enables tissue repair and induces angiogenesis. It was hypothesized that the angiogenic effects of SWT are at least partly caused by enhanced macrophage recruitment. C57BL/6 mice were subjected to hind limb ischaemia with subsequent SWT or sham treatment. Muscles were analysed via immunofluorescence staining, reverse-transcription polymerase chain reaction and western blot. Gene expression and proteins involved in macrophage recruitment were analysed and tissue sections were stained for macrophages, including subsets, capillaries and arterioles. Laser Doppler perfusion imaging was performed to assess functional outcome. Treated muscles showed increased expression of the pivotal macrophage recruiting factor monocyte chemotactic protein 1 (MCP-1). Higher levels of macrophage marker CD14 were found. Increased numbers of macrophages after SWT could be confirmed by immunofluorescence staining. The expression of the M2 polarization promoting chemokine interleukin 13 was significantly elevated in the treatment group. Elevated mRNA expression of the M2 scavenger receptor CD163 was found after SWT. Immunofluorescence staining confirmed increased numbers of M2 macrophages after treatment. It was found that SWT resulted in higher number of capillaries and arterioles. Assessment of functional outcome revealed significantly improved limb perfusion in treated animals. Shock wave therapy causes increased macrophage recruitment and enhanced polarization towards reparative M2 macrophages in ischaemic muscle resulting in angiogenesis and improved limb perfusion and therefore represents a promising new treatment option for the treatment of ischaemic heart disease. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Ondas de Choque de Alta Energia , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Macrófagos/metabolismo , Perfusão , Animais , Arteríolas/patologia , Capilares/patologia , Contagem de Células , Polaridade Celular , Isquemia/patologia , Masculino , Camundongos Endogâmicos C57BL , Músculos/irrigação sanguínea , Músculos/patologiaRESUMO
Early and adequate restoration of endothelial and tubular renal function is a substantial step during regeneration after ischemia reperfusion (IR) injury, occurring, e.g., in kidney transplantation, renal surgery, and sepsis. While tubular epithelial cell injury has long been of central importance, recent perception includes the renal vascular endothelium. In this regard, the fibrin cleavage product fibrinopeptide Bß15-42 mitigate IR injury by stabilizing interendothelial junctions through its affinity to VE-cadherin. Therefore, this study focused on the effect of Bß15-42 on post-acute physiological renal regeneration. For this, adult male C57BL/6 mice were exposed to a 30 min bilateral renal ischemia and reperfusion for 24 h or 48 h. Animals were randomized in a non-operative control group, two operative groups each treated with i.v. administration of either saline or Bß15-42 (2.4 mg/kg) immediately prior to reperfusion. Endothelial activation and inflammatory response was attenuated in renal tissue homogenates by single application of Bß15-42. Meanwhile, Bß15-42 did not affect acute kidney injury markers. Regarding the angiogenetic players VEGF-A, Angiopoietin-1, Angiopoietin-2, however, we observed significant higher expressions at mRNA and trend to higher protein level in Bß15-42 treated mice, compared to saline treated mice after 48 h of IR, thus pointing toward an increased angiogenetic activity. Similar dynamics were observed for the intermediate filament vimentin, the cytoprotective protein klotho, stathmin and the proliferation cellular nuclear antigen, which were significantly up-regulated at the same points in time. These results suggest a beneficial effect of anatomical contiguously located endothelial cells on tubular regeneration through stabilization of endothelial integrity. Therefore, it seems that Bß15-42 represents a novel pharmacological approach in the targeted therapy of acute renal failure in everyday clinical practice.
RESUMO
Acute kidney injury remains an important cause of renal dysfunction. In this context, Toll-like receptors have been demonstrated to play a critical role in the induction of innate and inflammatory responses. Among these, Toll-like receptor 2 (TLR2) is constitutively expressed in tubular epithelial cells (TECs) of the kidney and is also known to mediate ischaemia reperfusion (IR) injury. Adult male C57BL/6JRj mice were randomized into seven groups (n = 8): a non-operative control group (CTRL) and six interventional groups in which mice were subjected to a 30 min. bilateral renal ischaemia. Immediately before reperfusion, mice were treated either with saline or with TLR2 antibody (clone T2.5) and harvested after ischaemia and reperfusion for 3, 24 and 48 hr. Analysed kidney homogenates of TLR2 antibody-treated mice displayed significantly decreased levels of TLR2 protein after 3 hr of IR compared to saline-treated mice. Accordingly, the degree of AKT phosphorylation was significantly decreased after 3 hr of IR compared to saline-treated animals. TUNEL staining revealed significantly higher apoptosis rates in TLR2 antibody-treated animals compared to saline-treated mice after 3 and 24 hr of IR. Further, a positive correlation between TLR2 protein expression and phosphorylation of AKT as well as a negative correlation with the number of TUNEL-positive cells could be observed. Inhibition of TLR2 and its signalling pathway by a single application of TLR2 antibody results in reduced phosphorylation of AKT and consecutively increased apoptosis.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Rim/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/induzido quimicamente , Receptor 2 Toll-Like/antagonistas & inibidores , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/patologia , Animais , Anticorpos/toxicidade , Modelos Animais de Doenças , Rim/enzimologia , Rim/imunologia , Rim/patologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Fosforilação , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismoRESUMO
Nitro-fatty acids (NFAs) are endogenously occurring lipid mediators exerting strong anti-inflammatory effects and acting as anti-oxidants in a number of animal models of inflammation. These NFA effects are mediated by targeting important regulatory proteins involved in inflammatory processes, such as 5-lipoxygenase, soluble epoxide hydrolase, or NF-κB. In the present study, we investigated the anti-tumorigenic effects of NFAs on colorectal cancer (CRC) cells in cell culture-based experiments and in a murine xenograft model of human CRC. We could show that 9-NOA suppresses the viability of CRC cells (HCT-116 and HT-29) by inducing a caspase-dependent apoptosis via the intrinsic apoptotic pathway. Co-treatment with the pan-caspase inhibitor Q-VD-OPH counteracted the NFA-mediated apoptosis in both cell lines. Furthermore, NFAs affected the cell cycle transition and reduced the oxygen consumption rate (OCR) immediately. On the contrary to their well-known anti-oxidative properties, NFAs mediated the generation of mitochondrial oxidative stress in human CRC cells. Additionally, similar to the cytostatic drug mitomycin, 9-NOA significantly reduced tumor growth in a murine xenograft model of human colorectal cancer. In contrast to the established cytostatic drug, 9-NOA treatment was well tolerated by mice. This study delivers a novel mechanistic approach for nitro-fatty acid-induced inhibition of CRC cell growth by targeting mitochondrial functions such as the mitochondrial membrane potential and mitochondrial respiration. We suggest these naturally occurring lipid mediators as a new class of well tolerated chemotherapeutic drug candidates for treatment of CRC or potentially other inflammation-driven cancer types.
Assuntos
Anti-Inflamatórios/metabolismo , Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácidos Graxos/farmacologia , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacosRESUMO
AIMS: 5-Lipoxygenase (5-LO) is the key enzyme of leukotriene (LT) biosynthesis and is critically involved in a number of inflammatory diseases such as arthritis, gout, bronchial asthma, atherosclerosis, and cancer. Because 5-LO contains critical nucleophilic amino acids, which are sensitive to electrophilic modifications, we determined the consequences of a drug-mediated intracellular release of nitric oxide (NO) on 5-LO product formation by human granulocytes and on 5-LO-dependent pulmonary inflammation in vivo. RESULTS: Clinically relevant concentrations of NO-releasing nonsteroidal anti-inflammatory drugs and other agents releasing NO intracellularly suppress 5-LO product synthesis in isolated human granulocytes via direct S-nitrosylation of 5-LO at the catalytically important cysteines 416 and 418. Furthermore, suppression of 5-LO product formation was observed in ionophore-stimulated human whole blood and in an animal model of pulmonary inflammation. INNOVATION: Here, we report for the first time that drugs releasing NO intracellularly are efficient 5-LO inhibitors in vitro and in vivo at least equivalent to approved 5-LO inhibitors. CONCLUSION: Our findings provide a novel mechanistic strategy for the development of a new class of drugs suppressing LT biosynthesis by site-directed nitrosylation. The results may also help to better understand the well-recognized anti-inflammatory clinically relevant actions of NO-releasing drugs. Furthermore, our study describes in detail a novel molecular mode of action of NO. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Angel Lanas, Hartmut Kühn, Joan Clària, Orina Belton. Antioxid. Redox Signal. 28, 1265-1285.