RESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease and lupus nephritis is a major risk factor for morbidity and mortality. Notch-3 signaling induced by membrane-bound or soluble ligands such as YB-1 constitutes an evolutionarily conserved pathway that determines major decisions in cell fate. Mass spectrometry of extracellular YB-1 in sera from patients with SLE and lupus-prone mice revealed specific post-translational guanidinylation of two lysine residues within the highly conserved cold-shock domain of YB-1 (YB-1-G). These modifications highly correlated with SLE disease activity, especially in patients with lupus nephritis and resulted in enhanced activation of Notch-3 signaling in T lymphocytes. The importance of YB-1:Notch-3 interaction in T cells was further evidenced by increased interleukin (Il)10 expression following YB-1-G stimulation and detection of both, YB-1-G and Notch-3, in kidneys of MRL.lpr mice by mass spectrometry imaging. Notch-3 expression and activation was significantly up-regulated in kidneys of 20-week-old MRL.lpr mice. Notably, lupus-prone mice with constitutional Notch-3 depletion (B6.Faslpr/lprNotch3-/-) exhibited an aggravated lupus phenotype with significantly increased mortality, enlarged lymphoid organs and aggravated nephritis. Additionally, these mice displayed fewer regulatory T cells and reduced amounts of anti-inflammatory IL-10. Thus, our results indicate that the YB-1:Notch-3 axis exerts protective effects in SLE and that Notch-3 deficiency exacerbates the SLE phenotype.
Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Receptor Notch3/metabolismo , Fatores de Transcrição/metabolismo , Animais , Humanos , Lúpus Eritematoso Sistêmico/complicações , Camundongos , Camundongos Endogâmicos MRL lpr , Transdução de Sinais , Linfócitos T ReguladoresRESUMO
Acute kidney injury is a common complication of advanced liver disease and increased mortality of these patients. Here, we analyzed the role of Y-box protein-1 (YB-1), a nucleic acid binding protein, in the bile duct ligation model of liver fibrosis and monitored liver and subsequent kidney damage. Following bile duct ligation, both serum levels of liver enzymes and expression of hepatic extracellular matrix components such as type I collagen were significantly reduced in mice with half-maximal YB-1 expression (Yb1+/-) as compared to their wild-type littermates. By contrast, expression of the chemokine CXCL1 was significantly augmented in these Yb1+/- mice. YB-1 was identified as a potent transcriptional repressor of the Cxcl1 gene. Precision-cut kidney slices from Yb1+/- mice revealed higher expression of the CXCL1 receptor CXCR2 as well as enhanced responsivity to CXCL1 compared to those from wild-type mice. Increased CXCL1 content in Yb1+/- mice led to pronounced bile duct ligation-induced damage of the kidneys monitored as parameters of tubular epithelial injury and immune cell infiltration. Pharmacological blockade of CXCR2 as well as application of an inhibitory anti-CXCL1 antibody significantly mitigated early systemic effects on the kidneys following bile duct ligation whereas it had only a modest impact on hepatic inflammation and function. Thus, our analyses provide direct evidence that YB-1 crucially contributes to hepatic fibrosis and modulates liver-kidney crosstalk by maintaining tight control over chemokine CXCL1 expression.
Assuntos
Cirrose Hepática , Ácidos Nucleicos , Fatores de Transcrição , Animais , Proteínas de Transporte , Rim , Ligadura , Fígado/patologia , Cirrose Hepática/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. In vitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.
Assuntos
Hipertensão/patologia , Rim/patologia , Linfocinas/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Angiotensinogênio/metabolismo , Animais , Pressão Sanguínea/genética , Células Cultivadas , Colágeno Tipo IV/genética , Modelos Animais de Doenças , Fibrose , Hepatócitos , Humanos , Hipertensão/etiologia , Hipertensão/genética , Linfocinas/antagonistas & inibidores , Masculino , Camundongos , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Cultura Primária de Células , Regulação para Cima , Ureter/cirurgiaRESUMO
Complement factor C5a has two known receptors, C5aR, which mediates proinflammatory effects, and C5L2, a potential C5a decoy receptor. We previously identified C5a/C5aR signaling as a potent profibrotic pathway in the kidney. Here we tested for the first time the role of C5L2 in renal fibrosis. In unilateral ureteral obstruction (UUO)-induced kidney fibrosis, the expression of C5aR and C5L2 increased similarly and gradually as fibrosis progressed and was particularly prominent in injured dilated tubules. Genetic deficiency of either C5aR or C5L2 significantly reduced UUO-induced tubular injury. Expression of key proinflammatory mediators, however, significantly increased in C5L2- compared with C5aR-deficient mice, but this had no effect on the number of renal infiltrating macrophages or T cells. Moreover, in C5L2-/- mice, the cytokine and matrix metalloproteinase-inhibitor tissue inhibitor of matrix metalloproteinase-1 was specifically enhanced. Consequently, in C5L2-/- mice the degree of renal fibrosis was similar to wild type (WT), albeit with reduced mRNA expression of some fibrosis-related genes. In contrast, C5aR-/- mice had significantly reduced renal fibrosis compared with WT and C5L2-/- mice in UUO. In vitro experiments with primary tubular cells demonstrated that deficiency for either C5aR or C5L2 led to a significantly reduced expression of tubular injury and fibrosis markers. Vice versa, stimulation of WT tubular cells with C5a significantly induced the expression of these markers, whereas the absence of either receptor abolished this induction. In conclusion, in experimental renal fibrosis C5L2 and C5aR both contribute to tubular injury, and, while C5aR acts profibrotic, C5L2 does not play a role in extracellular matrix accumulation, arguing against C5L2 functioning simply as a decoy receptor.
Assuntos
Complemento C5a/metabolismo , Fibrose/imunologia , Nefropatias/imunologia , Receptores de Quimiocinas/metabolismo , Animais , Complemento C5a/imunologia , Fibrose/genética , Rim/imunologia , Rim/patologia , Nefropatias/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologiaRESUMO
Renal fibrosis is a common underlying process of progressive kidney diseases. We investigated the role of macrophage migration inhibitory factor (MIF), a pleiotropic proinflammatory cytokine, in this process. In mice subjected to unilateral ureteral obstruction, genetic deletion or pharmacologic inhibition of MIF aggravated fibrosis and inflammation, whereas treatment with recombinant MIF was beneficial, even in established fibrosis. In two other models of progressive kidney disease, global Mif deletion or MIF inhibition also worsened fibrosis and inflammation and associated with worse kidney function. Renal MIF expression was reduced in tubular cells in fibrotic compared with healthy murine and human kidneys. Bone marrow chimeras showed that Mif expression in bone marrow-derived cells did not affect fibrosis and inflammation after UUO. However, Mif gene deletion restricted to renal tubular epithelial cells aggravated these effects. In LPS-stimulated tubular cell cultures, Mif deletion led to enhanced G2/M cell-cycle arrest and increased expression of the CDK inhibitor 1B (p27Kip1) and of proinflammatory and profibrotic mediators. Furthermore, MIF inhibition reduced tubular cell proliferation in vitro In all three in vivo models, global Mif deletion or MIF inhibition caused similar effects and attenuated the expression of cyclin B1 in tubular cells. Mif deletion also resulted in reduced tubular cell apoptosis after UUO. Recombinant MIF exerted opposing effects on tubular cells in vitro and in vivo Our data identify renal tubular MIF as an endogenous renoprotective factor in progressive kidney diseases, raising the possibility of pharmacologic intervention with MIF pathway agonists, which are in advanced preclinical development.
Assuntos
Ciclo Celular , Inflamação/patologia , Oxirredutases Intramoleculares/metabolismo , Túbulos Renais/citologia , Rim/patologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Animais , Apoptose , Proliferação de Células , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Fibrose/patologia , Humanos , Rim/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Traumatismo por Reperfusão , Fator de Crescimento Transformador beta/metabolismoRESUMO
The Y-box-binding protein (YB)-1 plays a non-redundant role in both systemic and local inflammatory response. We analysed YB-1-mediated expression of the immune regulatory cytokine IL-10 in both LPS and sterile inflammation induced by unilateral renal ischaemia-reperfusion (I/R) and found an important role of YB-1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis-regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB-1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB-1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL-10 mRNA expression in mice following LPS challenge and in I/R. Mice with half-maximal expression of YB-1 (Yb1+/- ) had diminished IL-10 expression upon LPS challenge. In I/R, Yb1+/- mice exhibited ameliorated kidney injury/inflammation in the early-phase (days 1 and 5), however showed aggravated long-term damage (day 21) with increased expression of IL-10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context-specific decisions concerning YB-1 function and that a fine-tuning of YB-1, for example, via a post-translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.
Assuntos
Regulação da Expressão Gênica , Interleucina-10/genética , Rim/metabolismo , RNA Mensageiro/genética , Traumatismo por Reperfusão/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Éxons , Heterozigoto , Homozigoto , Inflamação , Interleucina-10/metabolismo , Íntrons , Rim/patologia , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
We have identified platelet-derived growth factor (PDGF)-CC as a potent profibrotic mediator in kidney fibrosis and pro-angiogenic mediator in glomeruli. Because renal fibrosis is associated with progressive capillary rarefaction, we asked whether PDGF-CC neutralization in fibrosis might have detrimental anti-angiogenic effects leading to aggravated peritubular capillary loss. We analyzed capillary rarefaction in mice with and without PDGF-CC neutralization (using genetically deficient mice and neutralizing antibodies), in three different models of renal interstitial fibrosis, unilateral ureteral obstruction, unilateral ischemia-reperfusion, Col4a3-deficient (Alport) mice, and healthy animals. Independent of the effect of PDGF-CC neutralization on renal fibrosis, we found no difference in capillary rarefaction between PDGF-CC-neutralized mice and mice with intact PDGF-CC. We also found no differences in microvascular leakage (determined by extravasation of Evans Blue Dye) and in renal relative blood volume quantified using in vivo microcomputed tomography. PDGF-CC neutralization had no effects on renal microvasculature in healthy animals. Capillary endothelium did not express PDGF receptor-α, suggesting that potential PDGF-CC effects would have to be indirect. PDGF-CC neutralization or deficiency was not associated with preservation or accelerated loss of peritubular capillaries, suggesting no significant pro-angiogenic effects of PDGF-CC during renal fibrosis. From a clinical perspective, the profibrotic effects of PDGF-CC outweigh the pro-angiogenic effects and, thus, do not limit a potential therapeutic use of PDGF-CC inhibition in renal fibrosis.
Assuntos
Capilares/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Linfocinas/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Capilares/patologia , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Rim/patologia , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Linfocinas/genética , Camundongos , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
PURPOSE: To assess the apparent diffusion coefficient (ADC) derived from diffusion-weighted (DW) magnetic resonance imaging (MRI) as a specific marker of renal fibrosis in rats with unilateral ureteral obstruction (UUO). MATERIALS AND METHODS: Thirteen rats were analyzed in group 1 (n = 4), group 2 (n = 3), and group 3 (n = 6) and measured using a clinical 3.0T MR scanner. Groups 1 and 2 were used to establish the final imaging protocols for group 3. DW imaging with four b-values (0, 50, 300, 800 s/mm(2) ) was conducted before UUO, at days 3 and 5 after UUO, after release of the obstruction, and after sacrifice. Renal cortical ADCs were correlated with histological and ultrastructural analyses. RESULTS: ADC values of group 3 are shown as mean ± standard deviation of [10(-3) mm(2) /s]. On day 5, in vivo cortical ADC of obstructed fibrotic kidneys was significantly reduced compared to unobstructed kidneys (1.4 ± 0.086 vs. 1.535 ± 0.087, P = 0.0018). Postmortem ADC dropped by 50% and was significantly increased in obstructed vs. unobstructed kidneys (0.711 ± 0.094 vs. 0.566 ± 0.049, P = 0.0046). Histopathology of obstructed kidneys showed tubular dilation, tubular cell atrophy, and expansion of the interstitial space. Postmortem ADC correlated tightly with tubular lumen area (r = 0.9, P < 0.001), fibronectin (r = 0.8, P = 0.003), collagen type I (r = 0.73, P = 0.007), and interstitial expansion (r = 0.69, P = 0.013). CONCLUSION: Compared to the in vivo measurements, postmortem renal ADCs were considerably reduced and, unlike in vivo, fibrotic kidneys exhibited consistently higher ADC compared to healthy kidney parenchyma. Our data suggest that in vivo ADC is unlikely to be a direct measure of renal fibrosis.
Assuntos
Imagem de Difusão por Ressonância Magnética/métodos , Modelos Animais de Doenças , Interpretação de Imagem Assistida por Computador/métodos , Rim/patologia , Animais , Fibrose , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Platelet-derived growth factors (PDGF) are key mediators of organ fibrosis. We investigated whether PDGF-C(-/-) mice or mice treated with neutralizing PDGF-C antibodies are protected from bile duct ligation-induced liver fibrosis, and we compared the effects with those of PDGF-C deficiency or neutralization on kidney fibrosis induced by unilateral ureteral obstruction. Unexpectedly, and in contrast to kidney fibrosis, PDGF-C deficiency or antagonism did not protect from liver fibrosis or functional liver impairment. Furthermore, the hepatic infiltration of monocytes/macrophages/dendritic cells and chemokine mRNA expression (CC chemokine ligand [CCL]5, CCL2, and CC chemokine receptor 2 [CCR2]) remained unchanged. Transcript expression of PDGF ligands increased in both liver and kidney fibrosis and was not affected by neutralization of PDGF-C. In kidney fibrosis, PDGF-C deficiency or antagonism led to reduced expression and signaling of PDGF-receptor (R)-α- and PDGFR-ß-chains. In contrast, in liver fibrosis there was either no difference (PDGF-C(-/-) mice) or even an upregulation of PDGFR-ß and signaling (anti-PDGF-C group). Finally, in vitro studies in portal myofibroblasts pointed to a predominant role of PDGF-B and PDGF-D signaling in liver fibrosis. In conclusion, our study revealed significant differences between kidney and liver fibrosis in that PDGF-C mediates kidney fibrosis, whereas antagonism of PDGF-C in liver fibrosis appears to be counteracted by significant upregulation and increased PDGFR-ß signaling. PDGF-C antagonism, therefore, may not be effective to treat liver fibrosis.
Assuntos
Rim/patologia , Cirrose Hepática/metabolismo , Linfocinas/fisiologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Cirrose Hepática/etiologia , Cirrose Hepática/prevenção & controle , Linfocinas/antagonistas & inibidores , Linfocinas/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/deficiência , Ratos , Ratos Sprague-Dawley , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Obstrução Ureteral/complicaçõesRESUMO
Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.
RESUMO
In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [NOV]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor-ß, and fibronectin, and increased PDGF-receptor-α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes.
Assuntos
Glomerulonefrite Membranoproliferativa/metabolismo , Neovascularização Fisiológica/fisiologia , Proteína Sobre-Expressa em Nefroblastoma/fisiologia , Actinas/metabolismo , Doença Aguda , Indutores da Angiogênese/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Progressão da Doença , Eletroquimioterapia/métodos , Células Endoteliais/efeitos dos fármacos , Fibronectinas/metabolismo , Terapia Genética/métodos , Mesângio Glomerular/patologia , Glomerulonefrite Membranoproliferativa/patologia , Glomerulonefrite Membranoproliferativa/fisiopatologia , Glomerulonefrite Membranoproliferativa/terapia , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/efeitos dos fármacos , Masculino , Células Mesangiais/patologia , Músculo Esquelético/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/sangue , Proteína Sobre-Expressa em Nefroblastoma/genética , Proteína Sobre-Expressa em Nefroblastoma/farmacologia , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Platelet-derived growth factors (PDGF)-AA and -CC mediate renal fibroblast proliferation and/or renal fibrosis. Whereas PDGF-CC binds to both the PDGF receptors (PDGFRs)-αα- and -αß, PDGF-AA binds more selectively to the αα-receptor, suggesting potential differences in the biological activities. METHODS: We compared signal transduction, gene expression as well as changes in the proteome induced by PDGF-AA and -CC in rat renal fibroblasts, which express both PDGFR subunits. The growth factor concentrations used were chosen based on their equipotency in inducing rat renal fibroblast proliferation. RESULTS: Both PDGF-AA and PDGF-CC induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Renal fibroblast proliferation induced by either PDGF-AA or -CC could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase (MAPK)-, Janus-kinase (JAK)/signal transducers and activators of transcription (STAT) and phosphatidyl-inositol-3-kinase (PI3K) pathway, pointing to the involvement of all the three pathways. However, quantitative differences between both the stimulations were minor. Additive or synergistic effects by stimulating simultaneously with PDGF-AA and -CC were not observed. Using a proteomic approach we found eleven differentially expressed proteins, which were quantitatively altered after treatment with either PDGF-AA or PDGF-CC. The regulation of calreticulin and inorganic pyrophosphatase 1 could be verified by western blotting. CONCLUSIONS: PDGF-AA and -CC exhibit almost identical biological effects on signal transduction and proteome in cultured renal fibroblasts, suggesting that the ligands exert their activity essentially through the commonly bound PDGFR-αα. Nonetheless, two differentially expressed proteins were identified which might be involved in the development of renal failure.
Assuntos
Fibroblastos/metabolismo , Rim/metabolismo , Linfocinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Eletroforese em Gel Bidimensional , Ensaio de Desvio de Mobilidade Eletroforética , Fibroblastos/citologia , Rim/citologia , Linfocinas/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação , Fator de Crescimento Derivado de Plaquetas/genética , Proteômica , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Several molecular changes in colorectal adenomas provide the basis of the adenoma-carcinoma sequence. We investigated the expression of xenobiotic ATP-binding cassette (ABC) transporters in humans and in ApcMin mice and conducted functional studies estimating the importance of the expression changes. Twenty-nine adenomas from 21 patients and eight adenomas from four ApcMin mice were analyzed using Western blotting and quantitative Real-time polymerase chain reaction (RT-PCR). Adjacent healthy tissue served as control for each polyp. Breast cancer resistance protein (BCRP) was significantly downregulated in human colorectal adenomas (to 28 ± 35% of adjacent healthy tissue). This was in line with data from ApcMin mice adenomas, where downregulation was significant as well (to 58 ± 34%). In parallel, quantitative RT-PCR showed BCRP mRNA downregulation in human adenomas (to 17 ± 31%). Basal multidrug resistance-associated protein 2 expression was low and did not change in adenomas; multidrug resistance transporter 1 expression also did not differ between adenomas and healthy tissue. In a functional study, ApcMin mice received radioactively labelled 2-amino-1-methyl-6-phenylimidazo[4,5-ß] pyridine (PhIP), a food colon carcinogen and substrate of BCRP, by oral gavage with analysis of PhIP accumulation and DNA adduct formation 48 hr later. In this setting, we could demonstrate a higher carcinogen concentration in adenomas of ApcMin mice (181 ± 113% of normal tissue) including immunohistochemical detection of PhIP-DNA adducts. We conclude that significant transcriptional downregulation of BCRP/Bcrp leads to higher carcinogen concentrations in colorectal adenomas of mice and men. This might promote the adenoma-carcinoma sequence by higher genotoxic effects. The results indicate a possible role of transporter deficiencies in susceptibility for colon carcinoma.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenoma/metabolismo , Neoplasias do Colo/metabolismo , Imidazóis/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Carcinógenos , Adutos de DNA/análise , Regulação para Baixo , Feminino , Alimentos , Humanos , Masculino , Camundongos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , RNA Mensageiro/análise , XenobióticosRESUMO
Recent studies have shown renoprotective effects of the peroxisome proliferator-activated receptor-α (PPAR-α), but its role in kidney fibrosis is unknown. In order to gain insight into this, we examined the effect of a novel PPAR-α agonist, BAY PP1, in two rat models of renal fibrosis: unilateral ureteral obstruction and the 5/6 nephrectomy. In healthy animals, PPAR-α was expressed in tubular but not in interstitial cells. Upon induction of fibrosis, PPAR-α was significantly downregulated, and treatment with BAY PP1 significantly restored its expression. During ureteral obstruction, treatment with BAY PP1 significantly reduced tubulointerstitial fibrosis, proliferation of interstitial fibroblasts, and TGF-ß(1) expression. Treatment with a less potent PPAR-α agonist, fenofibrate, had no effects. Treatment with BAY PP1, initiated in established disease in the 5/6 nephrectomy, halted the decline of renal function and significantly ameliorated renal fibrosis. In vitro, BAY PP1 had no direct effect on renal fibroblasts but reduced collagen, fibronectin, and TGF-ß(1) expression in tubular cells. Conditioned media of BAY PP1-treated tubular cells reduced fibroblast proliferation. Thus, renal fibrosis is characterized by a reduction of PPAR-α expression, and treatment with BAY PP1 restores PPAR-α expression and ameliorates renal fibrosis by modulating the cross-talk between tubular cells and fibroblasts. Hence, potent PPAR-α agonists might be useful in the treatment of renal fibrosis.
Assuntos
Ácido 3-Mercaptopropiônico/análogos & derivados , Fibrose/prevenção & controle , Nefropatias/tratamento farmacológico , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Pirimidinas/uso terapêutico , Ácido 3-Mercaptopropiônico/farmacologia , Ácido 3-Mercaptopropiônico/uso terapêutico , Animais , Proliferação de Células/efeitos dos fármacos , Fibrose/tratamento farmacológico , Nefropatias/patologia , Túbulos Renais/patologia , Nefrectomia , Substâncias Protetoras , Pirimidinas/farmacologia , Ratos , Resultado do Tratamento , Obstrução Ureteral/tratamento farmacológicoRESUMO
Chronic HCV (hepatitis C virus)-associated cirrhosis represents a major indication for liver transplantation. Bile acids contribute to hepatic stellate cell activation as a key event in fibrogenesis. The aim of the present study was to investigate the role of bile acids and polymorphisms in bile acid level-regulating genes on fibrosis progression. A total of 206 subjects with chronic HCV infection were included for ABCB11 (ATP-binding cassette, subfamily B, member II) 1331T>C and NR1H4 (nuclear receptor) -1G>T genotyping, 178 of which were analysed for fibrosis stage. Exclusion criteria were HBV (hepatitis B virus) or HIV coinfection, alcohol >40 g/day and morbid obesity. A total of 358 patients with NAFLD (non-alcoholic fatty liver disease) were genotyped for comparison with a non-viral liver disease. Caucasian individuals (n = 110), undergoing liver resection for focal hepatic metastasis, served as controls. The ABCB11 1331C allele was significantly overrepresented in HCV patients compared with controls {allelic frequency 62.9%; OR (odds ratio), 1.41 [95% CI (confidence interval), 1.012-1.965]}. Median plasma bile acid levels were not significantly increased in the CC compared with TT genotype [7.2 (1-110) µmol/l compared with 3.5 (1-61) µmol/l; values are medians (range). A significant association between the presence of cirrhosis and ABCB11 genotype (CC compared with CT or TT, P=0.047) was observed in the χ2 test and independent of other risk factors of age, gender, body mass index and disease duration in multivariate analysis (P = 0.010). No such association could be observed in fatty liver patients with regard to advanced fibrosis (F ≥ 2). The common ABCB11 1331CC genotype, which is present in 40% of HCV patients and renders the carrier susceptible to increased bile acid levels, is associated with cirrhosis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Hepatite C Crônica/genética , Cirrose Hepática/genética , Polimorfismo de Nucleotídeo Único , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácidos e Sais Biliares/sangue , Progressão da Doença , Métodos Epidemiológicos , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Feminino , Predisposição Genética para Doença , Genótipo , Hepatite C Crônica/sangue , Hepatite C Crônica/complicações , Humanos , Cirrose Hepática/sangue , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.
Assuntos
Caquexia/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Insuficiência Renal Crônica/metabolismo , Síndrome de Emaciação/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Ativinas/genética , Ativinas/metabolismo , Animais , Caquexia/etiologia , Caquexia/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Atrofia Muscular/etiologia , Atrofia Muscular/genética , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Síndrome de Emaciação/etiologia , Síndrome de Emaciação/genéticaRESUMO
The nuclear bile acid receptor FXR (farnesoid-X-receptor) has recently been implicated in the pathophysiology of non-alcoholic fatty liver disease because selective FXR-agonists improve glucose and lipid metabolism in rodent models of obesity. However, the regulation of FXR and other relevant nuclear receptors as well as their lipogenic target genes in fatty liver is still not revealed in detail. Livers were harvested from 14-week-old male ob/ob mice and wild-type controls. Serum bile acids were quantified by radioimmunoassay. mRNA and protein expression of transporters and nuclear receptors was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting, whereas DNA binding to the IR-1 element was examined by electrophoretic mobility shift assay. In this study we show: (i) bile acid retention in ob/ob mice, (ii) a resulting FXR upregulation and binding to the IR-1 element in ob/ob animals and (iii) concomitant activation of the fatty acid synthase as a potential lipogenic FXR target gene in vivo. The present study suggests a potential role of hepatic bile acid retention and FXR activation in the induction of lipogenic target genes. Differences between intestinal and hepatic FXR could explain apparent contradictory information regarding its effects on fatty liver disease.
Assuntos
Ácidos e Sais Biliares/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Regulação para CimaRESUMO
DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear-mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box. Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase.
Assuntos
DNA Ligases/química , DNA Ligases/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Núcleo Celular/enzimologia , Clonagem Molecular , Códon de Iniciação/genética , Sequência Conservada , DNA Ligase Dependente de ATP , DNA Ligases/genética , Técnica Indireta de Fluorescência para Anticorpo , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , TemperaturaRESUMO
Cdc37 is a molecular chaperone that interacts with a range of clients and co-chaperones, forming various high molecular mass complexes. Cdc37 sequence homology among species is low. High homology between yeast and metazoan proteins is restricted to the extreme N-terminal region, which is known to bind clients that are predominantly protein kinases. We show that despite the low homology, both Saccharomyces cerevisiae and human Cdc37 are able to substitute for the Schizosaccharomyces pombe protein in a strain deleted for the endogenous cdc37 gene. Expression of a construct consisting of only the N-terminal domain of S. pombe Cdc37, lacking the postulated heat-shock protein (Hsp) 90-binding and homodimerization domains, can also sustain cellular viability, indicating that Cdc37 dimerization and interactions with the cochaperone Hsp90 may not be essential for Cdc37 function in S. pombe. Biochemical investigations showed that a small proportion of total cellular Cdc37 occurs in a high molecular mass complex that also contains Hsp90. These data indicate that the N-terminal domain of Cdc37 carries out essential functions independently of the Hsp90-binding domain and dimerization of the chaperone itself.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Drosophila/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/fisiologia , Schizosaccharomyces/citologia , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Chaperoninas , Cromatografia em Gel , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Humanos , Imunoprecipitação , Chaperonas Moleculares/química , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases.