RESUMEN
Fibrosis is a common denominator of several pathological conditions. Over the last decade, Calreticulin has emerged as a critical player in the fibrotic processes in many tissues and organs. Here we review the recent advances in our understanding of the regulatory roles of Calreticulin in renal fibrosis. In particular, a proteomic screen that we performed more than 15 years ago, for the identification of novel components involved in the mechanisms of renal fibrosis, led to the observation that Calreticulin is associated with the initiation and progression of kidney fibrosis in a rodent model. We also showed that altered expression levels of Calreticulin in vitro and in vivo are significantly affecting the fibrotic phenotype in cellular systems and animal models, respectively. We also identified an upstream regulatory mechanism that mediates the transcriptional control of Calreticulin expression during the progression of renal fibrosis, by showing that the druggable orphan nuclear receptor NR5A2 and its SUMOylation is involved in this action. These data provide novel targets for future pharmacological interventions against fibrosis. In addition, further proteomic analysis uncovered a correlation between the up-regulation of Calreticulin and that of 14-3-3σ protein. Collectively, our previous observations suggest that Calreticulin is a central node in a regulatory axis that controls the initiation and progression of renal fibrosis.
Asunto(s)
Calreticulina , Enfermedades Renales , Animales , Calreticulina/genética , Calreticulina/metabolismo , Proteómica , Fibrosis , Enfermedades Renales/genética , Enfermedades Renales/patología , Regulación de la Expresión Génica , Riñón/patologíaRESUMEN
Chronic kidney disease, the end result of most renal and some systemic diseases, is a common condition where renal function is compromised due to fibrosis. During renal fibrosis, calreticulin, a multifunctional chaperone of the endoplasmic reticulum (ER) is up-regulated in tubular epithelial cells (TECs) both in vitro and in vivo. Proteomic analysis of cultured TECs overexpressing calreticulin led to the identification of the family of 14-3-3 proteins as key proteins overexpressed as well. Furthermore, an increased expression in the majority of 14-3-3 family members was observed in 3 different animal models of renal pathologies: the unilateral ureteric obstruction, the nephrotoxic serum administration and the ischaemia-reperfusion. In all these models, the 14-3-3σ isoform (also known as stratifin) was predominantly overexpressed. As in all these models ischaemia is a common denominator, we showed that the ischaemia-induced transcription factor HIF1α is specifically associated with the promoter region of the 14-3-3σ gene. Finally, we evaluated the expression of the family of 14-3-3 proteins and specifically 14-3-3σ in biopsies from IgA nephropathy and membranous nephropathy patients. These results propose an involvement of 14-3-3σ in renal pathology and provide evidence for the first time that hypoxia may be responsible for its altered expression.
Asunto(s)
Proteínas 14-3-3/genética , Biomarcadores de Tumor/genética , Exorribonucleasas/genética , Glomerulonefritis por IGA/genética , Glomerulonefritis Membranosa/genética , Insuficiencia Renal Crónica/genética , Daño por Reperfusión/genética , Obstrucción Ureteral/genética , Proteínas 14-3-3/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Exorribonucleasas/metabolismo , Fibrosis , Regulación de la Expresión Génica , Glomerulonefritis por IGA/metabolismo , Glomerulonefritis por IGA/patología , Glomerulonefritis Membranosa/metabolismo , Glomerulonefritis Membranosa/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Proteómica/métodos , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Starting from 1994, every 2 years, an international workshop is organized focused on calreticulin and other endoplasmic reticulum chaperones. In 2017, the workshop took place at Delphi Greece. Participants from North and South America, Europe, Asia and Australia presented their recent data and discussed them extensively with their colleagues. Presentations dealt with structural aspects of calreticulin and calnexin, the role of Ca2+ in cellular signalling and in autophagy, the endoplasmic reticulum stress and the unfolded protein response, the role of calreticulin in immune responses. Several presentations focused on the role of calreticulin and other ER chaperones in a variety of disease states, including haemophilia, obesity, diabetes, Sjogren's syndrome, Chagas diseases, multiple sclerosis, amyotrophic lateral sclerosis, neurological malignancies (especially glioblastoma), haematological malignancies (especially essential thrombocythemia and myelofibrosis), lung adenocarcinoma, renal pathology with emphasis in fibrosis and drug toxicity. In addition, the role of calreticulin and calnexin in growth and wound healing was discussed, as well as the possible use of extracellular calreticulin as a marker for certain diseases. It was agreed that the 13th International Calreticulin Workshop will be organized in 2019 in Montreal, Quebec, Canada.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Calreticulina/genética , Retículo Endoplásmico/genética , Hemofilia A/genética , Neoplasias/genética , Obesidad/genética , Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/patología , Animales , Autofagia , Calcio/metabolismo , Calnexina/genética , Calnexina/aislamiento & purificación , Calreticulina/inmunología , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica , Hemofilia A/inmunología , Hemofilia A/patología , Humanos , Inmunidad Innata , Chaperonas Moleculares/genética , Chaperonas Moleculares/inmunología , Neoplasias/inmunología , Neoplasias/patología , Obesidad/inmunología , Obesidad/patología , Transducción de Señal , Respuesta de Proteína Desplegada , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunologíaRESUMEN
Renal fibrosis is the common anatomical feature underlying the progression of chronic kidney disease, a leading cause of morbidity and mortality worldwide. In a previous study, we demonstrated that during development of renal fibrosis in a rat model of unilateral ureteric obstruction, calreticulin (CRT) is up-regulated in tubular epithelial cells (TECs). In the present study, we used in vitro and in vivo approaches to examine the role of CRT in TECs and its contribution to the progression of fibrosis. In cultured renal TECs, CRT overexpression induced acquisition of an altered, profibrotic cellular phenotype. Consistently, the opposite effects were observed for CRT knockdown. Subsequently, we confirmed that critical changes observed in vitro were also apparent in tubular cells in vivo in the animal model of unilateral ureteric obstruction. In agreement with these results, we demonstrate that substantial (50%) reduction in the expression of CRT reduced the development of tubulointerstitial fibrosis at a comparable level through regulation of inflammation, transcriptional activation, transforming growth factor ß1-associated effects, and apoptosis. In summary, our findings establish that CRT is critically involved in the molecular mechanisms that drive renal fibrosis progression and indicate that inhibition of CRT expression might be a therapeutic target for reduction of fibrosis and chronic kidney disease development.
Asunto(s)
Calreticulina/metabolismo , Células Epiteliales/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Regulación hacia Arriba , Animales , Apoptosis , Biomarcadores/metabolismo , Línea Celular , Movimiento Celular , Proliferación Celular , Colágeno/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Estrés del Retículo Endoplásmico , Células Epiteliales/patología , Fibrosis , Técnicas de Silenciamiento del Gen , Heterocigoto , Humanos , Masculino , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Ratas , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Secreted wingless-interacting protein (Swim) is the Drosophila ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen like 1 (TINAGL1), also known as lipocalin-7 (LCN7), or adrenocortical zonation factor 1 (AZ-1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In Drosophila, Swim was initially identified in the eggshell matrix and was subsequently detected in the culture medium of S2 cells. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation, together with RNAi-knockdown studies, suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wg-signalling in Drosophila, because flies without Swim are viable and fertile. Here, we use the UAS/Gal4 expression system together with confocal imaging to analyze the in vivo localization of a chimeric Swim-GFP in the developing Drosophila embryo. Our data fully support the notion that Swim is an extracellular matrix component that is secreted upon ectopic expression and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). Interestingly, the accumulation of Swim at the MAS does not require integrins. In conclusion, Swim is an extracellular matrix component, and Swim may exhibit overlapping functions in concert with other undefined components.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Mamíferos , Transducción de Señal/fisiologíaRESUMEN
Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well-established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up-regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up-regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin-induced) and heart fibrosis (desmin-null) were examined. In the lung model, calreticulin expression was up-regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF-beta, a well known major contributing factor in several fibrotic processes, was found to up-regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.
Asunto(s)
Calreticulina/metabolismo , Fibrosis/genética , Fibrosis/metabolismo , Regulación de la Expresión Génica , Fibrosis Pulmonar/metabolismo , Animales , Bleomicina/toxicidad , Calreticulina/genética , Línea Celular Transformada , Células Cultivadas , Colágeno/biosíntesis , Desmina/genética , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Biológicos , Proteómica/métodos , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Ratas , Ratas Wistar , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Pulmonary fibrosis is a common feature of a large group of lung diseases. The molecular mechanisms underlying pulmonary fibrosis and the key macromolecules involved are not fully understood yet. In an effort to better understand aspects of pulmonary fibrosis, the established bleomycin injection model in mice was used and the focus of the present study was on integrin-linked kinase (ILK) expression. ILK is an intracellular protein involved in the regulation of integrin-mediated processes. In fibrosis, ILK has been examined in the kidney and in the liver where it mediates epithelial to mesenchymal transition (EMT) and hepatic stellate cell activation, respectively. However, information on ILK's involvement in lung fibrosis is missing. In order to examine ILK's role in pulmonary fibrosis, we used both an in vivo and an in vitro approach. In vivo, the bleomycin model was used in order to examine ILK's expression and localization in the fibrotic lung. In vitro, transforming growth factor-ß1 was used to induce fibrotic characteristics and EMT in alveolar epithelial cells. ILK's role in alveolar EMT was studied by siRNA. Our results demonstrate that in the animal model used, ILK exhibits a decrease in expression at early stages of the fibrotic process and that a specific subset of fibroblasts is expressing ILK. The in vitro experiments suggested that ILK is not directly involved in E-cadherin downregulation and initiation of EMT (as is the case in renal fibrosis) but is involved in upregulation of vimentin. These results suggest that ILK is involved in lung fibrosis in a tissue-specific manner and raise the possibility to use it as a specific therapeutic target for lung fibrosis in the future.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/enzimología , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/patología , ARN Interferente Pequeño , Transducción de Señal/fisiologíaRESUMEN
Tubulointerstitial nephritis antigen (TIN-Ag) is a recently described basement membrane glycoprotein reactive with autoantibodies in some forms of immunologically mediated human tubulointerstitial nephritis. This report presents the complete cDNA and predicted amino acid sequences of two human TIN-Ag mRNA species referred to as TIN1 and TIN2. Translation through the open reading frames of these clones indicates the presence of a signal peptide and putative pre-propeptide. TIN1 additionally contains a characteristic laminin-like epidermal growth factor (EGF) motif and significant homology within the carboxy terminus with the cysteine proteinase family of enzymes. The EGF motif bears important similarities in the positions of cysteines with two motifs in the propeptide of von Willebrand factor. The EGF motif and part of the region that is homologous with the cysteine proteinase family are removed from the TIN2 cDNA. However, the rest of the sequence is identical in these two forms, indicating an alternatively spliced TIN-Ag mRNA product. Both forms contain putative calcium-binding sites. Secondary structure predictions strongly suggest differences between TIN1 and TIN2 leading to the hypothesis that these two forms of TIN-Ag may exhibit differences in their function. Expression studies with appropriate probes demonstrate expression mainly in the kidney and in the intestinal epithelium and lack of expression in other tissues. In the kidney, both TIN1 and TIN2 transcripts are detected, however, TIN1 appears to be the predominant form.