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1.
Kidney Int ; 97(4): 741-752, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32061437

RESUMEN

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.


Asunto(s)
Cirrosis Hepática , Ácidos Nucleicos , Factores de Transcripción , Animales , Proteínas Portadoras , Riñón , Ligadura , Hígado/patología , Cirrosis Hepática/genética , Ratones , Ratones Endogámicos C57BL
2.
Kidney Int ; 97(2): 289-303, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31882173

RESUMEN

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.


Asunto(s)
Lupus Eritematoso Sistémico , Nefritis Lúpica , Receptor Notch3/metabolismo , Factores de Transcripción/metabolismo , Animales , Humanos , Lupus Eritematoso Sistémico/complicaciones , Ratones , Ratones Endogámicos MRL lpr , Transducción de Señal , Linfocitos T Reguladores
3.
Kidney Int ; 95(5): 1103-1119, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30827511

RESUMEN

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.


Asunto(s)
Hipertensión/patología , Riñón/patología , Linfocinas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Angiotensinógeno/metabolismo , Animales , Presión Sanguínea/genética , Células Cultivadas , Colágeno Tipo IV/genética , Modelos Animales de Enfermedad , Fibrosis , Hepatocitos , Humanos , Hipertensión/etiología , Hipertensión/genética , Linfocinas/antagonistas & inhibidores , Masculino , Ratones , Ratones Noqueados , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Cultivo Primario de Células , Regulación hacia Arriba , Uréter/cirugía
4.
Am J Physiol Renal Physiol ; 314(1): F35-F46, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-28903945

RESUMEN

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.


Asunto(s)
Complemento C5a/metabolismo , Fibrosis/inmunología , Enfermedades Renales/inmunología , Receptores de Quimiocina/metabolismo , Animales , Complemento C5a/inmunología , Fibrosis/genética , Riñón/inmunología , Riñón/patología , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología
5.
J Am Soc Nephrol ; 28(12): 3590-3604, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28801314

RESUMEN

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.


Asunto(s)
Ciclo Celular , Inflamación/patología , Oxidorreductasas Intramoleculares/metabolismo , Túbulos Renales/citología , Riñón/patología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Animales , Apoptosis , Proliferación Celular , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Femenino , Fibrosis/patología , Humanos , Riñón/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Daño por Reperfusión , Factor de Crecimiento Transformador beta/metabolismo
6.
J Cell Mol Med ; 21(12): 3494-3505, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28664613

RESUMEN

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.


Asunto(s)
Regulación de la Expresión Génica , Interleucina-10/genética , Riñón/metabolismo , ARN Mensajero/genética , Daño por Reperfusión/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Exones , Heterocigoto , Homocigoto , Inflamación , Interleucina-10/metabolismo , Intrones , Riñón/patología , Lipopolisacáridos , Masculino , Ratones , Ratones Transgénicos , Unión Proteica , ARN Mensajero/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Factores de Transcripción/metabolismo
7.
Am J Pathol ; 185(8): 2132-42, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26216283

RESUMEN

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.


Asunto(s)
Capilares/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Linfocinas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Capilares/patología , Modelos Animales de Enfermedad , Fibrosis/metabolismo , Fibrosis/patología , Riñón/patología , Enfermedades Renales/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Linfocinas/genética , Ratones , Ratones Noqueados , Factor de Crecimiento Derivado de Plaquetas/genética , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología
8.
J Magn Reson Imaging ; 42(4): 990-8, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25630829

RESUMEN

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.


Asunto(s)
Imagen de Difusión por Resonancia Magnética/métodos , Modelos Animales de Enfermedad , Interpretación de Imagen Asistida por Computador/métodos , Riñón/patología , Animales , Fibrosis , Masculino , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
9.
Am J Pathol ; 182(1): 107-17, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23141925

RESUMEN

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.


Asunto(s)
Riñón/patología , Cirrosis Hepática/metabolismo , Linfocinas/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Cirrosis Hepática/etiología , Cirrosis Hepática/prevención & control , Linfocinas/antagonistas & inhibidores , Linfocinas/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miofibroblastos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/deficiencia , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Obstrucción Ureteral/complicaciones
10.
iScience ; 27(3): 109255, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38444605

RESUMEN

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.

11.
Sci Transl Med ; 16(735): eadi1501, 2024 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-38381845

RESUMEN

Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), for which therapeutic options are limited. Strategies to promote intestinal tissue tolerance during aGVHD may improve patient outcomes. Using single-cell RNA sequencing, we identified a lipocalin-2 (LCN2)-expressing neutrophil population in mice with intestinal aGVHD. Transfer of LCN2-overexpressing neutrophils or treatment with recombinant LCN2 reduced aGVHD severity, whereas the lack of epithelial or hematopoietic LCN2 enhanced aGVHD severity and caused microbiome alterations. Mechanistically, LCN2 induced insulin-like growth factor 1 receptor (IGF-1R) signaling in macrophages through the LCN2 receptor SLC22A17, which increased interleukin-10 (IL-10) production and reduced major histocompatibility complex class II (MHCII) expression. Transfer of LCN2-pretreated macrophages reduced aGVHD severity but did not reduce graft-versus-leukemia effects. Furthermore, LCN2 expression correlated with IL-10 expression in intestinal biopsies in multiple cohorts of patients with aGVHD, and LCN2 induced IGF-1R signaling in human macrophages. Collectively, we identified a LCN2-expressing intestinal neutrophil population that reduced aGVHD severity by decreasing MHCII expression and increasing IL-10 production in macrophages. This work provides the foundation for administration of LCN2 as a therapeutic approach for aGVHD.


Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Humanos , Animales , Ratones , Neutrófilos/patología , Interleucina-10 , Lipocalina 2/genética , Enfermedad Injerto contra Huésped/genética , Macrófagos/patología , Enfermedad Aguda
12.
Biol Chem ; 394(12): 1623-36, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24084358

RESUMEN

Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells.


Asunto(s)
Aneuploidia , Diferenciación Celular , Hipocampo/citología , Células-Madre Neurales/citología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo
13.
Am J Pathol ; 180(5): 1979-90, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22538190

RESUMEN

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.


Asunto(s)
Glomerulonefritis Membranoproliferativa/metabolismo , Neovascularización Fisiológica/fisiología , Proteína Hiperexpresada del Nefroblastoma/fisiología , Actinas/metabolismo , Enfermedad Aguda , Inductores de la Angiogénesis/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Progresión de la Enfermedad , Electroquimioterapia/métodos , Células Endoteliales/efectos de los fármacos , Fibronectinas/metabolismo , Terapia Genética/métodos , Mesangio Glomerular/patología , Glomerulonefritis Membranoproliferativa/patología , Glomerulonefritis Membranoproliferativa/fisiopatología , Glomerulonefritis Membranoproliferativa/terapia , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/efectos de los fármacos , Masculino , Células Mesangiales/patología , Músculo Esquelético/metabolismo , Proteína Hiperexpresada del Nefroblastoma/sangre , Proteína Hiperexpresada del Nefroblastoma/genética , Proteína Hiperexpresada del Nefroblastoma/farmacología , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Proteínas Recombinantes/farmacología
14.
Langmuir ; 29(12): 4057-67, 2013 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-23445373

RESUMEN

We measure silane density and Sulfo-EMCS cross-linker coupling efficiency on aminosilane films by high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) measurements. We then characterize DNA immobilization and hybridization on these films by (32)P-radiometry. We find that the silane film structure controls the efficiency of the subsequent steps toward DNA hybridization. A self-limited silane monolayer produced from 3-aminopropyldimethylethoxysilane (APDMES) provides a silane surface density of ~3 nm(-2). Thin (1 h deposition) and thick (19 h deposition) multilayer films are generated from 3-aminopropyltriethoxysilane (APTES), resulting in surfaces with increased roughness compared to the APDMES monolayer. Increased silane surface density is estimated for the 19 h APTES film, due to a ∼32% increase in surface area compared to the APDMES monolayer. High cross-linker coupling efficiencies are measured for all three silane films. DNA immobilization densities are similar for the APDMES monolayer and 1 h APTES. However, the DNA immobilization density is double for the 19 h APTES, suggesting that increased surface area allows for a higher probe attachment. The APDMES monolayer has the lowest DNA target density and hybridization efficiency. This is attributed to the steric hindrance as the random packing limit is approached for DNA double helices (dsDNA, diameter ≥ 2 nm) on a plane. The heterogeneity and roughness of the APTES films reduce this steric hindrance and allow for tighter packing of DNA double helices, resulting in higher hybridization densities and efficiencies. The low steric hindrance of the thin, one to two layer APTES film provides the highest hybridization efficiency of nearly 88%, with 0.21 dsDNA/nm(2). The XPS data also reveal water on the cross-linker-treated surface that is implicated in device aging.


Asunto(s)
ADN/química , Propilaminas/química , Silanos/química , Dióxido de Silicio/química , Reactivos de Enlaces Cruzados/química , ADN/síntesis química , Microscopía de Fuerza Atómica , Hibridación de Ácido Nucleico , Radioisótopos de Fósforo , Espectroscopía de Fotoelectrones , Radiometría , Succinimidas/química , Propiedades de Superficie , Agua
15.
Nephrol Dial Transplant ; 28(4): 889-900, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23229927

RESUMEN

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.


Asunto(s)
Fibroblastos/metabolismo , Riñón/metabolismo , Linfocinas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Electroforesis en Gel Bidimensional , Ensayo de Cambio de Movilidad Electroforética , Fibroblastos/citología , Riñón/citología , Linfocinas/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/genética , Proteómica , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
16.
Int J Cancer ; 129(3): 546-52, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21544799

RESUMEN

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.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenoma/metabolismo , Neoplasias del Colon/metabolismo , Imidazoles/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Carcinógenos , Aductos de ADN/análisis , Regulación hacia Abajo , Femenino , Alimentos , Humanos , Masculino , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Mensajero/análisis , Xenobióticos
17.
Kidney Int ; 80(11): 1182-97, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21814170

RESUMEN

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.


Asunto(s)
Ácido 3-Mercaptopropiónico/análogos & derivados , Fibrosis/prevención & control , Enfermedades Renales/tratamiento farmacológico , Receptores Activados del Proliferador del Peroxisoma/agonistas , Pirimidinas/uso terapéutico , Ácido 3-Mercaptopropiónico/farmacología , Ácido 3-Mercaptopropiónico/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Fibrosis/tratamiento farmacológico , Enfermedades Renales/patología , Túbulos Renales/patología , Nefrectomía , Sustancias Protectoras , Pirimidinas/farmacología , Ratas , Resultado del Tratamiento , Obstrucción Ureteral/tratamiento farmacológico
18.
Clin Sci (Lond) ; 120(7): 287-96, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20883210

RESUMEN

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.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Hepatitis C Crónica/genética , Cirrosis Hepática/genética , Polimorfismo de Nucleótido Simple , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Adulto , Anciano , Anciano de 80 o más Años , Ácidos y Sales Biliares/sangre , Progresión de la Enfermedad , Métodos Epidemiológicos , Hígado Graso/sangre , Hígado Graso/genética , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Hepatitis C Crónica/sangre , Hepatitis C Crónica/complicaciones , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Adulto Joven
19.
J Clin Invest ; 131(11)2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-34060483

RESUMEN

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.


Asunto(s)
Caquexia/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Insuficiencia Renal Crónica/metabolismo , Síndrome Debilitante/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Activinas/genética , Activinas/metabolismo , Animales , Caquexia/etiología , Caquexia/genética , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Atrofia Muscular/etiología , Atrofia Muscular/genética , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/genética , Síndrome Debilitante/etiología , Síndrome Debilitante/genética
20.
Biol Chem ; 391(12): 1441-9, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20868235

RESUMEN

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.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Hígado Graso/etiología , Hígado Graso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Regulación hacia Arriba
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