RESUMO
Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.
Assuntos
Fígado/enzimologia , Pró-Proteína Convertase 9 , Proteinúria , Receptores de LDL , Animais , Heparitina Sulfato , Proteinúria/induzido quimicamente , Ratos , Receptores de LDL/genética , SubtilisinasRESUMO
Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation.
Assuntos
Anticorpos/imunologia , Rejeição de Enxerto/diagnóstico , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Células Matadoras Naturais/imunologia , Adulto , Idoso , Aloenxertos/citologia , Aloenxertos/imunologia , Aloenxertos/patologia , Biomarcadores/análise , Biópsia , Estudos de Casos e Controles , Biologia Computacional , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Rim/citologia , Rim/imunologia , Rim/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Prognóstico , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Renal fibrogenesis is associated with increased ECM remodeling and release of collagen fragments in urine in progressive renal disease. We investigated the diagnostic value of urinary collagen degradation products in a proteinuria-driven fibrosis rat model with and without anti-fibrotic S1P-receptor modulator FTY720 treatment. METHODS: Proteinuria was induced in male Wistar rats by Adriamycin (ADR) injection (n = 16). Healthy rats served as controls (n = 12). Six weeks post-injection, all underwent renal biopsy, and FTY720-treatment started in ADR-rats (n = 8) and controls (n = 6). Others remained untreated. Rats were sacrificed after 12 weeks. Collagen type I (C1M) and III (C3M) degradation fragments were measured in blood and urine using ELISA. Kidneys were stained for various inflammatory and fibrotic markers. RESULTS: Six weeks post-injection proteinuria increased (versus controls, P < 0.001) and although no accumulation of interstitial renal collagen type III (iColl3) was observed at this time, urinary C3M (uC3M) and C1M (uC1M) were significantly increased (both P < 0.001). At 12 weeks, uC3M (P < 0.001) and uC1M (P < 0.01) further increased in ADR-rats versus controls, just as fibronectin, PDGF-ß receptor, hyaluronan (all P < 0.01), iColl3, PAS, myofibroblasts, macrophages and T-cells (all P < 0.05). FTY720-treatment reduced accumulation of immune cells, α-SMA+ myofibroblasts and PAS-score, but not iColl3 and uC3M. Correlation analyses indicated that uC3M and uC1M reflected and predicted tubulointerstitial fibrogenesis. CONCLUSIONS: These data displayed urinary collagen breakdown products as sensitive early markers of interstitial fibrosis, preceding histological fibrotic changes, which might replace the invasive renal biopsy procedure to assess fibrosis. Anti-fibrotic FTY720 intervention reduced some fibrotic markers without affecting collagen type III metabolism.
Assuntos
Colágeno Tipo III/urina , Colágeno Tipo I/urina , Progressão da Doença , Rim/patologia , Animais , Biomarcadores/sangue , Biomarcadores/urina , Colágeno Tipo I/sangue , Colágeno Tipo III/sangue , Doxorrubicina , Fibrose , Cloridrato de Fingolimode/farmacologia , Masculino , Proteinúria/induzido quimicamente , Proteinúria/complicações , Proteinúria/patologia , Proteólise , Ratos WistarRESUMO
Renal fibrosis leads to end-stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN-γ is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN-γ causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell-specific delivery of IFN-γ to platelet-derived growth factor receptor ß (PDGFRß)-expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN-γ conjugated to PDGFRß-recognizing peptide [(PPB)-polyethylene glycol (PEG)-IFN-γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN-γ. PDGFRß expression was >3-fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with α-smooth muscle actin-positive (SMA(+)) myofibroblasts. In vitro, PPB-PEG-IFN-γ significantly inhibited col1a1, col1a2, and α-SMA mRNA expression in TGF-ß-activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB-PEG-IFN-γ specifically accumulated in PDGFRß-positive myofibroblasts. PPB-PEG-IFN-γ treatment significantly reduced renal collagen I, fibronectin, and α-SMA mRNA and protein expression. Compared with vehicle treatment, PPB-PEG-IFN-γ preserved tubular morphology, reduced interstitial T-cell infiltration, and attenuated lymphangiogenesis (all P < 0.05) without affecting peritubular capillary density. PPB-PEG-IFN-γ reduced IFN-γ-related side effects as manifested by reduced major histocompatibility complex class II expression in brain tissue (P < 0.05 vs. free IFN-γ). Our findings demonstrate that specific targeting of IFN-γ to PDGFRß-expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects.
Assuntos
Encéfalo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Fibrose/tratamento farmacológico , Interferon-alfa/farmacologia , Nefropatias/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibrose/metabolismo , Fibrose/patologia , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Nefropatias/metabolismo , Nefropatias/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Células NIH 3T3 , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hydrogen sulfide (H2S), carbon monoxide (CO) and nitric oxide (NO) share signaling and vasorelaxant properties and are involved in proliferation and apoptosis. Inhibiting NO production or availability induces hypertension and proteinuria, which is prevented by concomitant blockade of the H2S producing enzyme cystathionine γ-lyase (CSE) by d,l-propargylglycine (PAG). We hypothesized that blocking H2S production ameliorates Angiotensin II (AngII)-induced hypertension and renal injury in a rodent model. Effects of concomitant administration of PAG or saline were therefore studied in healthy (CON) and AngII hypertensive rats. In CON rats, PAG did not affect systolic blood pressure (SBP), but slightly increased proteinuria. In AngII rats PAG reduced SBP, proteinuria and plasma creatinine (180 ± 12 vs. 211 ± 19 mmHg; 66 ± 35 vs. 346 ± 92 mg/24 h; 24 ± 6 vs. 47 ± 15 µmol/L, respectively; p < 0.01). Unexpectedly, kidney to body weight ratio was increased in all groups by PAG (p < 0.05). Renal injury induced by AngII was reduced by PAG (p < 0.001). HO-1 gene expression was increased by PAG alone (p < 0.05). PAG increased inner cortical tubular cell proliferation after 1 week and decreased outer cortical tubular nucleus number/field after 4 weeks. In vitro proximal tubular cell size increased after exposure to PAG. In summary, blocking H2S production with PAG reduced SBP and renal injury in AngII infused rats. Independent of the cardiovascular and renal effects, PAG increased HO-1 gene expression and kidney weight. PAG alone increased tubular cell size and proliferation in-vivo and in-vitro. Our results are indicative of a complex interplay of gasotransmitter signaling/action of mutually compensatory nature in the kidney.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Alcinos/farmacologia , Angiotensina II/efeitos adversos , Pressão Sanguínea/efeitos dos fármacos , Glicina/análogos & derivados , Sulfeto de Hidrogênio/metabolismo , Alcinos/administração & dosagem , Animais , Proliferação de Células , Glicina/administração & dosagem , Glicina/farmacologia , Heme Oxigenase-1/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Óxido Nítrico , Tamanho do Órgão , Ratos , Ratos Sprague-DawleyRESUMO
Lymphatic vessels (LVs) are involved in a number of physiological and pathophysiological processes such as fluid homoeostasis, immune surveillance, and resolution of inflammation and wound healing. Lymphangiogenesis, the outgrowth of existing LVs and the formation of new ones, has received increasing attention over the past decade on account of its prominence in organ physiology and pathology, which has been enabled by the development of specific tools to study lymph vessel functions. Several studies have been devoted to renal lymphatic vasculature and lymphangiogenesis in kidney diseases, such as chronic renal transplant dysfunction, primary renal fibrotic disorders, proteinuria, diabetic nephropathy and renal inflammation. This review describes the most recent findings on lymphangiogenesis, with a specific focus on renal lymphangiogenesis and its impact on renal diseases. We suggest renal lymphatics as a possible target for therapeutic interventions in renal medicine to dampen tubulointerstitial tissue remodelling and improve renal functioning.
Assuntos
Inflamação/fisiopatologia , Linfangiogênese , Vasos Linfáticos/fisiopatologia , Insuficiência Renal Crônica/fisiopatologia , Humanos , Transplante de Rim/efeitos adversos , Cicatrização/fisiologiaRESUMO
Staphylococcus aureus is considered a high priority pathogen by the World Health Organization due to its high prevalence and the potential to form biofilms. Currently, the available treatments for S. aureus biofilm-associated infections do not target the extracellular polymeric substances (EPS) matrix. This matrix is a physical barrier to bactericidal agents, contributing to the increase of antimicrobial tolerance. The present work proposes the development of lipid nanoparticles encapsulating caspofungin (CAS) as a matrix-disruptive nanosystem. The nanoparticles were functionalized with D-amino acids to target the matrix. In a multi-target nano-strategy against S. aureus biofilms, CAS-loaded nanoparticles were combined with a moxifloxacin-loaded nanosystem, as an adjuvant to promote the EPS matrix disruption. In vitro and in vivo studies showed biofilm reduction after combining the two nanosystems. Besides, the combinatory therapy showed no signs of bacterial dissemination into vital organs of mice, while dissemination was observed for the treatment with the free compounds. Additionally, the in vivo biodistribution of the two nanosystems revealed their potential to reach and accumulate in the biofilm region, after intraperitoneal administration. Thus, this nano-strategy based on the encapsulation of matrix-disruptive and antibacterial agents is a promising approach to fight S. aureus biofilms.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Camundongos , Distribuição Tecidual , Biofilmes , Infecções Estafilocócicas/microbiologia , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
BACKGROUND: Recent experimental findings demonstrate vascular endothelial growth factor C (VEGF-C)-mediated water-free storage of salt in the interstitium, which prevents a salt-sensitive blood pressure state. It is unknown whether this mechanism plays a role in salt homeostasis and regulation of blood pressure in humans as well. Therefore, we investigated circulating VEGF-C levels and blood pressure during different well-controlled salt intake in chronic kidney disease (CKD) patients and in healthy subjects. METHODS: In two crossover studies, non-diabetic proteinuric CKD patients (n = 32) and healthy subjects (n = 31) were treated with consecutively a low-sodium diet (LS, aim 50 mmol Na(+)/day) and a high-sodium diet (HS, aim 200 mmol Na(+)/day) in random order, during two 6-week (CKD patients) and two 1-week periods (healthy subjects). RESULTS: We found that VEGF-C levels are higher during HS than during LS in CKD patients (P = 0.034) with a trend towards higher VEGF-C in healthy subjects as well (P = 0.070). In CKD patients, HS was associated with higher NT-proBNP levels (P = 0.005) and body weight (P = 0.013), consistent with extracellular volume (ECV) expansion and with higher blood pressure (P < 0.001), indicating salt sensitivity. In healthy subjects, blood pressure was not affected by dietary salt (P = 0.14), despite a rise in ECV (P = 0.023). DISCUSSION: Our findings support a role for VEGF-C-mediated salt homeostasis in humans. Considering the salt sensitivity of blood pressure, this buffering mechanism appears to be insufficient in proteinuric CKD patients. Future studies are needed to provide causality and to substantiate the clinical and therapeutic relevance of this VEGF-C regulatory mechanism in humans.
Assuntos
Dieta Hipossódica , Homeostase/efeitos dos fármacos , Hipertensão , Falência Renal Crônica/sangue , Proteinúria/sangue , Cloreto de Sódio na Dieta/administração & dosagem , Fator C de Crescimento do Endotélio Vascular/sangue , Adulto , Peso Corporal , Estudos de Casos e Controles , Estudos Cross-Over , Feminino , Humanos , Falência Renal Crônica/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteinúria/tratamento farmacológico , Proteinúria/etiologia , Adulto JovemRESUMO
Objectives: Chronic kidney disease (CKD), accompanied by renal dysfunction, fibrosis, and apoptosis, is highly prevalent in postmenopausal women. We tested the hypothesis that isoflavone daidzein may ameliorate renal dysfunction and fibrosis through angiotensin II type 1 (AT1R) and angiotensin 1-7 (MasR) receptors in association with microRNAs 33a and 27a. Materials and Methods: Two weeks before the initiation of the experiments, rats (n=84) underwent ovariectomy (OVX). Then, unilateral ureteral obstruction (UUO) was performed in OVX rats, and animals were allocated to the following groups (n=21): sham vehicle (dimethyl sulfoxide; DMSO 1%), UUO vehicle, UUO+17ß-estradiol (E2), and UUO+daidzein. Each group encompassed three subgroups (n=7) treated with saline, A779 (MasR antagonist), or losartan (AT1R antagonist) for 15 days. The fractional urine excretion of sodium (FENa+) and potassium (FEK+), renal failure index (RFI), renal interstitial fibrosis (RIF index), glomerulosclerosis, miR-33a, and miR-27a expressions and their target genes were analyzed. Apoptosis was measured via cleaved caspase-3 immunohistochemistry. Results: UUO increased kidney weight, FENa+, FEK+, urine calcium, RFI, RIF index, glomerulosclerosis, and cleaved caspase-3. Moreover, expression of renal miR-33a and miR-27a, collagen3A1 mRNA, and protein were up-regulated post-UUO. Daidzein treatment alleviated the harmful effects of UUO especially in co-treatment with losartan. They also masked the anticipated worsening effects of A779 on UUO. Conclusion: Compared with E2, daidzein efficiently ameliorated renal dysfunction, fibrosis, and apoptosis through modulation of miR-33a and miR-27a expression and their crosstalk with AT1R and MasR. Therefore, daidzein might be a promising candidate for treating CKD in postmenopausal and older women.
RESUMO
Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.
Assuntos
Candida glabrata/enzimologia , Candida glabrata/genética , Proteínas Fúngicas/genética , Trato Gastrointestinal/microbiologia , Estresse Fisiológico/genética , Trealase/genética , Animais , Candida glabrata/efeitos dos fármacos , Candida glabrata/patogenicidade , Feminino , Proteínas Fúngicas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/genética , Células RAW 264.7 , Trealase/classificação , Trealase/metabolismo , VirulênciaRESUMO
BACKGROUND: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available. METHODS: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (Nâ¯=â¯117 and Nâ¯=â¯183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (Nâ¯=â¯387). This trial is registered with ClinicalTrials.gov, number NCT02832661. FINDINGS: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (Nâ¯=â¯49) and without (Nâ¯=â¯134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (Nâ¯=â¯41) and without antibody-mediated rejection (Nâ¯=â¯346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, pâ¯<â¯0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection. INTERPRETATION: We identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection. FUNDING: The Seventh Framework Programme (FP7) of the European Commission.
Assuntos
Anticorpos/imunologia , Biomarcadores , Ácidos Nucleicos Livres , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , RNA Mensageiro/genética , Adulto , Feminino , Rejeição de Enxerto/sangue , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , RNA Mensageiro/sangue , Curva ROC , Reprodutibilidade dos Testes , Transplante HomólogoRESUMO
Myofibroblasts are the key players in extracellular matrix remodeling, a core phenomenon in numerous devastating fibrotic diseases. Not only in organ fibrosis, but also the pivotal role of myofibroblasts in tumor progression, invasion and metastasis has recently been highlighted. Myofibroblast targeting has gained tremendous attention in order to inhibit the progression of incurable fibrotic diseases, or to limit the myofibroblast-induced tumor progression and metastasis. In this review, we outline the origin of myofibroblasts, their general characteristics and functions during fibrosis progression in three major organs: liver, kidneys and lungs as well as in cancer. We will then discuss the state-of-the art drug targeting technologies to myofibroblasts in context of the above-mentioned organs and tumor microenvironment. The overall objective of this review is therefore to advance our understanding in drug targeting to myofibroblasts, and concurrently identify opportunities and challenges for designing new strategies to develop novel diagnostics and therapeutics against fibrosis and cancer.
Assuntos
Antineoplásicos/farmacologia , Miofibroblastos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/química , Fibrose/tratamento farmacológico , Fibrose/patologia , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Miofibroblastos/patologiaRESUMO
BACKGROUND: High dietary sodium aggravates renal disease by affecting blood pressure and by its recently shown pro-inflammatory and pro-fibrotic effects. Moreover, pro-inflammatory modification of renal heparan sulfate (HS) can induce tissue remodeling. We aim to investigate if high sodium intake in normotensive rats converts renal HS into a pro-inflammatory phenotype, able to bind more sodium and orchestrate inflammation, fibrosis and lymphangiogenesis. METHODS: Wistar rats received a normal diet for 4 weeks, or 8% NaCl diet for 2 or 4 weeks. Blood pressure was monitored, and plasma, urine and tissue collected. Tissue sodium was measured by flame spectroscopy. Renal HS and tubulo-interstitial remodeling were studied by biochemical, immunohistochemical and qRT-PCR approaches. RESULTS: High sodium rats showed a transient increase in blood pressure (week 1; p<0.01) and increased sodium excretion (p<0.05) at 2 and 4 weeks compared to controls. Tubulo-interstitial T-cells, myofibroblasts and mRNA levels of VCAM1, TGF-ß1 and collagen type III significantly increased after 4 weeks (all p<0.05). There was a trend for increased macrophage infiltration and lymphangiogenesis (both p = 0.07). Despite increased dermal sodium over time (p<0.05), renal concentrations remained stable. Renal HS of high sodium rats showed increased sulfation (p = 0.05), increased L-selectin binding to HS (p<0,05), and a reduction of sulfation-sensitive anti-HS mAbs JM403 (p<0.001) and 10E4 (p<0.01). Hyaluronan expression increased under high salt conditions (p<0.01) without significant changes in the chondroitin sulfate proteoglycan versican. Statistical analyses showed that sodium-induced tissue remodeling responses partly correlated with observed HS changes. CONCLUSION: We show that high salt intake by healthy normotensive rats convert renal HS into high sulfated pro-inflammatory glycans involved in tissue remodeling events, but not in increased sodium storage.
Assuntos
Rim/metabolismo , Proteoglicanas/metabolismo , Sódio/farmacologia , Animais , Fibrose/metabolismo , Imunofluorescência , Ácido Hialurônico/metabolismo , Imuno-Histoquímica , Rim/efeitos dos fármacos , Linfangiogênese/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Pele/metabolismo , Cloreto de Sódio na Dieta/efeitos adversosRESUMO
Excessive lymphangiogenesis is associated with cancer progression and renal disease. Attenuation of lymphangiogenesis might represent a novel strategy to target disease progression although clinically approved anti-lymphangiogenic drugs are not available yet. VitaminD(VitD)-deficiency is associated with increased cancer risk and chronic kidney disease. Presently, effects of VitD on lymphangiogenesis are unknown. Given the apparently protective effects of VitD and the deleterious associations of lymphangiogenesis with renal disease, we here tested the hypothesis that VitD has direct anti-lymphangiogenic effects in vitro and is able to attenuate lymphangiogenesis in vivo. In vitro cultured mouse lymphatic endothelial cells (LECs) expressed VitD Receptor (VDR), both on mRNA and protein levels. Active VitD (calcitriol) blocked LEC tube formation, reduced LEC proliferation, and induced LEC apoptosis. siRNA-mediated VDR knock-down reversed the inhibitory effect of calcitriol on LEC tube formation, demonstrating how such inhibition is VDR-dependent. In vivo, proteinuric rats were treated with vehicle or paricalcitol for 6 consecutive weeks. Compared with vehicle-treated proteinuric rats, paricalcitol showed markedly reduced renal lymphangiogenesis. In conclusion, our data show that VitD is anti-lymphangiogenic through VDR-dependent anti-proliferative and pro-apoptotic mechanisms. Our findings highlight an important novel function of VitD demonstrating how it may have therapeutic value in diseases accompanied by pathological lymphangiogenesis.
Assuntos
Ergocalciferóis/farmacologia , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Proteinúria/tratamento farmacológico , Receptores de Calcitriol/genética , Vitamina D/farmacologia , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/toxicidade , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Camundongos , Proteinúria/induzido quimicamente , Proteinúria/metabolismo , Proteinúria/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Receptores de Calcitriol/antagonistas & inibidores , Receptores de Calcitriol/metabolismoRESUMO
BACKGROUND: Chronic renal transplant dysfunction is characterized by loss of renal function and tissue remodeling, including chronic inflammation and lymph vessel formation. Proteoglycans are known for their chemokine presenting capacity. We hypothesize that interruption of the lymphatic chemokine-proteoglycan interaction interferes with the lymphatic outflow of leukocytes from the renal graft and might decrease the anti-graft allo-immune response. METHODS: In a rat renal chronic transplant dysfunction model (female Dark-Agouti to male Wistar Furth), chemokines were profiled by qRT-PCR in microdissected tubulo-interstitial tissue. Disruption of lymphatic chemokine-proteoglycan interaction was studied by (non-anticoagulant) heparin-derived polysaccharides in vitro and in renal allografts. The renal allograft function was assessed by rise in plasma creatinine and urea. RESULTS: Within newly-formed lymph vessels of transplanted kidneys, numerous CD45+ leukocytes were found, mainly MHCII+, ED-1-, IDO-, HIS14-, CD103- antigen presenting cells, most likely representing a subset of dendritic cells. Treatment of transplanted rats with regular heparin and two different (non-)anticoagulant heparin derivatives revealed worsening of kidney function only in the glycol-split heparin treated group despite a two-fold reduction of tubulo-interstitial leukocytes (p<0.02). Quantitative digital image analysis however revealed increased numbers of intra-lymphatic antigen-presenting cells only in the glycol-split heparin group (p<0.01). The number of intra-lymphatic leukocytes significantly correlates with plasma creatinine and urea, and inversely with creatinine clearance. CONCLUSIONS: Treatment of transplanted rats with glycol-split heparin significantly increases the number of intra-lymphatic antigen presenting cells, by increased renal diffusion of lymphatic chemokines, thereby increasing the activation and recruitment of antigen presenting cells towards the lymph vessel. This effect is unwanted in the transplantation setting, but might be advantageous in e.g., dendritic cell vaccination.
Assuntos
Células Apresentadoras de Antígenos/citologia , Movimento Celular , Glicóis/química , Heparina/farmacologia , Transplante de Rim , Vasos Linfáticos/citologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Heparina/química , Imuno-Histoquímica , Masculino , Ratos , Ratos WistarRESUMO
Tissue fibrosis, characterized by excessive accumulation of aberrant extracellular matrix (ECM) produced by myofibroblasts, is a growing cause of mortality worldwide. Understanding the factors that induce myofibroblastic differentiation is paramount to prevent or reverse the fibrogenic process. Integrin-mediated interaction between the ECM and cytoskeleton promotes myofibroblast differentiation. In the present study, we explored the significance of integrin alpha 11 (ITGA11), the integrin alpha subunit that selectively binds to type I collagen during tissue fibrosis in the liver, lungs and kidneys. We showed that ITGA11 was co-localized with α-smooth muscle actin-positive myofibroblasts and was correlatively induced with increasing fibrogenesis in mouse models and human fibrotic organs. Furthermore, transcriptome and protein expression analysis revealed that ITGA11 knockdown in hepatic stellate cells (liver-specific myofibroblasts) markedly reduced transforming growth factor ß-induced differentiation and fibrotic parameters. Moreover, ITGA11 knockdown dramatically altered the myofibroblast phenotype, as indicated by the loss of protrusions, attenuated adhesion and migration, and impaired contractility of collagen I matrices. Furthermore, we demonstrated that ITGA11 was regulated by the hedgehog signaling pathway, and inhibition of the hedgehog pathway reduced ITGA11 expression and fibrotic parameters in human hepatic stellate cells in vitro, in liver fibrosis mouse model in vivo and in human liver slices ex vivo. Therefore, we speculated that ITGA11 might be involved in fibrogenic signaling and might act downstream of the hedgehog signaling pathway. These findings highlight the significance of the ITGA11 receptor as a highly promising therapeutic target in organ fibrosis.
Assuntos
Cadeias alfa de Integrinas/genética , Miofibroblastos/metabolismo , Fenótipo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Imuno-Histoquímica , Cadeias alfa de Integrinas/metabolismo , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Renal fibrosis cannot be adequately treated since anti-fibrotic treatment is lacking. Interferon-γ is a pro-inflammatory cytokine with anti-fibrotic properties. Clinical use of interferon-γ is hampered due to inflammation-mediated systemic side effects. We used an interferon-γ peptidomimetic (mimγ) lacking the extracellular IFNγReceptor recognition domain, and coupled it to the PDGFßR-recognizing peptide BiPPB. Here we tested the efficacy of mimγ-BiPPB (referred to as "Fibroferon") targeted to PDGFßR-overexpressing interstitial myofibroblasts to attenuate renal fibrosis without inducing inflammation-mediated side effects in the mouse unilateral ureter obstruction model.Unilateral ureter obstruction induced renal fibrosis characterized by significantly increased α-SMA, TGFß1, fibronectin, and collagens I and III protein and/or mRNA expression. Fibroferon treatment significantly reduced expression of these fibrotic markers. Compared to full-length IFNγ, anti-fibrotic effects of Fibroferon were more pronounced. Unilateral ureter obstruction-induced lymphangiogenesis was significantly reduced by Fibroferon but not full-length IFNγ. In contrast to full-length IFNγ, Fibroferon did not induce IFNγ-related side-effects as evidenced by preserved low-level brain MHC II expression (similar to vehicle), lowered plasma triglyceride levels, and improved weight gain after unilateral ureter obstruction.In conclusion, compared to full-length IFNγ, the IFNγ-peptidomimetic Fibroferon targeted to PDGFßR-overexpressing myofibroblasts attenuates renal fibrosis in the absence of IFNγ-mediated adverse effects.
Assuntos
Interferon gama/uso terapêutico , Rim/patologia , Linfangiogênese/efeitos dos fármacos , Miofibroblastos/metabolismo , Peptidomiméticos/uso terapêutico , Obstrução Ureteral/tratamento farmacológico , Animais , Matriz Extracelular/metabolismo , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
The sphingosine-1-phosphate (S1P) analog FTY720 exerts pleiotropic effects on the cardiovascular system and causes down-regulation of S1P receptors. Myogenic constriction is an important mechanism regulating resistance vessel function and is known to be modulated by S1P. Here we investigated myogenic constriction and vascular function of mesenteric arteries of rats chronically treated with FTY720. Wistar rats received FTY720 1mg/kg/daily for six weeks. At termination, blood pressure was recorded and small mesenteric arteries collected for vascular studies in a perfusion set up. Myogenic constriction to increased intraluminal pressure was low, but a sub-threshold dose of S1P profoundly augmented myogenic constriction in arteries of both controls and animals chronically treated with FTY720. Interestingly, endothelial denudation blocked the response to S1P in arteries of FTY720-treated animals, but not in control rats. In acute experiments, presence of FTY720 significantly augmented the contractile response to S1P, an effect that was partially abolished after the inhibition of cyclooxygenase (COX-)-derived prostaglandins. FTY720 down regulated S1P1 but not S1P2 in renal resistance arteries and in cultured human endothelial cells. This study therefore demonstrates the endothelium is able to compensate for the complete loss of responsiveness of the smooth muscle layer to S1P after long term FTY720 treatment through a mechanism that most likely involves enhanced production of contractile prostaglandins by the endothelium.
Assuntos
Células Endoteliais/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Lisofosfolipídeos/metabolismo , Artérias Mesentéricas/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Peso Corporal/efeitos dos fármacos , Células Endoteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Humanos , Masculino , Artérias Mesentéricas/fisiologia , Desenvolvimento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Pressão , Ratos , Ratos Wistar , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/metabolismo , Fatores de Tempo , Vasoconstrição/efeitos dos fármacosRESUMO
Some diseases associated with a temporary deterioration in kidney function and/or development of proteinuria show an apparently complete functional remission once the initiating trigger is removed. While it was earlier thought that a transient impairment of kidney function is harmless, accumulating evidence now suggests that these patients are more prone to developing renal failure later in life. We therefore sought to investigate to what extent renal functional changes, inflammation and collagen deposition are reversible after cessation of disease induction, potentially explaining residual sensitivity to damage. Using a rat model of Angiotensin II (Ang II)-induced hypertensive renal disease we show the development of severe hypertension (212 ± 10.43 vs. 146 ± 1.4 mmHg, p<0.001) and proteinuria (51.4 ± 6.3 vs. 14.7 ± 2.0 mg/24h, p<0.01) with declined creatinine clearance (2.0 ± 0.5 vs. 4.9 ± 0.6 mL/min, p<0.001) to occur after 3 weeks of Ang II infusion. At the structural level, Ang II infusion resulted in interstitial inflammation (18.8 ± 4.8 vs. 3.6 ± 0.5 number of macrophages, p<0.001), renal interstitial collagen deposition and lymphangiogenesis (4.1 ± 0.4 vs. 2.2 ± 0.4 number of lymph vessels, p<0.01). Eight weeks after cessation of Ang II, all clinical parameters, pre-fibrotic changes such as myofibroblast transformation and increase in lymph vessel number (lymphangiogenesis) returned to control values. However, glomerular desmin expression, glomerular and periglomerular macrophages and interstitial collagens remained elevated. These dormant abnormalities indicate that after transient renal function decline, inflammation and collagen deposition may persist despite normalization of the initiating pathophysiological stimulus perhaps rendering the kidney more vulnerable to further damage.