BET Proteins: An Approach to Future Therapies in Transplantation.

In order to develop new efficient therapies for organ transplantation, it is essential to acquire a comprehensive knowledge of the molecular mechanisms and processes, such as immune activation, chronic inflammation, and fibrosis, which lead to rejection and long-term graft loss. Recent efforts have shed some light on the epigenetic regulation associated with these processes. In this context, the bromo and extraterminal (BET) family of bromodomain proteins (BRD2, BRD3, BRD4, and BRDT) have emerged as major epigenetic players, connecting chromatin structure with gene expression changes. These proteins recognize acetylated lysines in histones and master transcription factors to recruit regulatory complex and, finally, modify the transcriptional program. Recent studies indicate that BET proteins are essential in the NF-kB-mediated inflammatory response, during the activation and differentiation of Th17-immune cells, and in profibrotic processes. Here, we review this new body of data and highlight the efficiency of BET inhibitors in several models of diseases. The promising results obtained from these preclinical models indicate that it may be time to translate these outcomes to the transplantation field, where epigenetics will be of increasing value in the coming years.

Conducting Action Research to Improve Operational Efficiency in Manufacturing: The Case of a First-Tier Automotive Supplier.

Optimising available resources and minimising production costs and throughput time is vital for first-tier suppliers in the worldwide automotive sector. To develop this type of optimisation and efficiency, MAHLE applied Action Research (AR) in one of its factories located in Spain. A multidisciplinary collaborative work team was created with the aim of deploying the AR initiative in combination with Lean Manufacturing and Six Sigma tools. Four improvement and learning cycles were deployed and key performance metrics were defined to collect and measure data in order to analyse the improvements achieved. The application of the AR initiative in the production line of a power filter device enabled improvements in both production times and quality indicators in the manufacturing process. The most outstanding results were the improvements made in the decrease in initial throughput time (34.78%) and in average daily rejections (73.53%). In addition, the AR initiative generated practical and theoretical contributions for business and academia, allowing the AR initiative to be applied in other areas of the company, and contributing to the current state of the art in the industrial application of this methodology.

[Connective tissue growth factor (CTGF): a key factor in the onset and progression of kidney damage]. / El factor de crecimiento de tejido conectivo (CTGF): factor clave en el inicio y la progresión del daño renal.

Connective tissue growth factor (CTGF) is increased in several pathologies associated with fibrosis, including multiple renal diseases. CTGF is involved in biological processes such as cell cycle regulation, migration, adhesion and angiogenesis. Its expression is regulated by various factors involved in renal damage, such as transforming growth factor- , Angiotensin II, high concentrations of glucose and cellular stress. CTGF is involved in the initiation and progression of renal damage to be able to induce an inflammatory response and promote fibrosis, identified as a potential therapeutic target in the treatment of kidney diseases. In this paper we review the main actions of CTGF in renal disease, the intracellular action mechanisms and therapeutic strategies for its blocking.

[Transcriptomics illustrate a deadly TRAIL to diabetic nephropathy]. / La transcriptómica ilustra nuevas vías letales en la nefropatía diabética.

Diabetic nephropathy is the most common cause of endstage renal disease. Approaches targeting angiotensin II significantly delay its progression. However, many patients still need renal replacement therapy. High throughput techniques such as unbiased gene expression profiling and proteomics may identify new therapeutic targets. Cell death is thought to contribute to progressive renal cell depletion in chronic nephropathies. A European collaborative effort recently applied renal biopsy transcriptomics to identify novel mediators of renal cell death in diabetic nephropathy. Twenty-five percent of cell death regulatory genes were upor downregulated in diabetic kidneys. TNF-related apoptosisinducing ligand (TRAIL) and osteoprotegerin had the highest level of expression. In diabetic nephropathy, tubular cells and podocytes express TRAIL. Inflammatory cytokines, including MIF via CD74, upregulate TRAIL. A high glucose environment sensitized renal cells to the lethal effect of TRAIL, while osteoprotegerin is protective. These results suggest that, in addition to glucose levels, inflammation and TRAIL are therapeutic targets in diabetic nephropathy.

Parathyroid hormone-related protein promotes inflammation in the kidney with an obstructed ureter.

Parathyroid hormone-related protein (PTHrP) promotes fibrogenesis in the acutely damaged kidney. Considering the relation between fibrosis and inflammation, we studied transgenic mice that overexpress PTHrP in the proximal tubule. When unilateral ureteric obstruction was induced in these transgenic mice, we found that they had more renal tubulointerstitial damage, leukocyte influx, and expression of proinflammatory factors than their control littermates. Reversal of PTHrP constitutive overexpression in these transgenic mice or treatment of control mice with the PTHrP antagonist (7-34) decreased this inflammatory response. Losartan, which abolished obstruction-induced endogenous PTHrP upregulation, also decreased the latter response but less effectively in transgenic mice. The PTHrP fragment (1-36) induced nuclear factor-kappaB (NF-kappaB) activation and proinflammatory cytokine overexpression in mouse cortical tubule cells in culture as well as migration of the macrophage cell line Raw 264.7. All these effects were decreased by PTHrP (7-34) and NF-kappaB or extracellular signal-regulated kinase (ERK) activation inhibitors. Our findings suggest a critical role of PTHrP in the renal inflammatory process that results from ureteral obstruction and indicate that ERK-mediated NF-kappaB activation seems to be an important mechanism whereby PTHrP triggers renal inflammation.

Gremlin: a novel mediator of epithelial mesenchymal transition and fibrosis in chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is the most frequent cause of chronic dysfunction and late loss of renal allografts. Epithelial mesenchymal transition (EMT) has been identified as responsible for the presence of activated interstitial fibroblasts (myofibroblasts) and transforming growth factor beta (TGF-beta)/Smad is the key signaling mediator. It has been proposed that the bone morphogenetic protein 7 (BMP-7) antagonist, Gremlin, could participate in EMT, as a downstream mediator of TGF-beta. METHODS: We evaluated 33 renal allograft biopsies, 16 of which showed CAN, versus 17 controls. By in situ hybridization we studied the expression of TGF-beta and Gremlin mRNA. Gremlin, BMP-7, E-cadherin, and alpha-smooth muscle actin (alpha-SMA) proteins were evaluated by immunohistochemistry and Smad3 activation by Southwestern. In cultured human tubuloepithelial cells (HK2 cell line), Gremlin induction by TGF-beta was studied by confocal microscopy. RESULTS: Among renal biopsies of transplanted patients with CAN, we detected up-regulation of TGF-beta in colocalization with Gremlin (RNA and protein), mainly in areas of tubulointerstitial fibrosis. In the same tubules, we observed decreased expression of E-cadherin and induction of vimentin and alpha-SMA. BMP-7 was significantly decreased in the CAN biopsies. In addition, HK2 stimulated with TGF-beta (1 ng/mL) induced Gremlin production at 72 hours. CONCLUSION: We postulated that Gremlin is a downstream mediator of TGF-beta, suggesting a role for Gremlin in EMT observed in CAN.

[Inflammation and atherothrombosis. Premature vascular injury. Diagnostic and therapeutic options for primary prevention]. / Inflamación y aterotrombosis. Precocidad de la lesión vascular. Opciones diagnósticas y terapéuticas para prevención primaria.

Atherosclerosis, an inflammatory disease, develops prematurely in CKD (Strength of Recommendation B). The performance of tests for the diagnosis of subclinical atherosclerosis and systemic inflammation should therefore be considered in these patients. - Patients with CKD are at high risk of vascular disease, implying the need for goal-based treatment of the vascular risk factors. - In this context, imaging tests for the diagnosis of subclinical atherosclerosis can be considered with the goal of individualizing treatment decisions on the management of cardiovascular risk factors when the patient is no longer receiving treatment consistent with the maximum risk level (Strength of Recommendation C). - The presence of subclinical atherosclerosis would raise cardiovascular risk to the maximum level (Strength of Recommendation C). - Carotid artery ultrasound with determination of carotid intima- media thickness (CIMT) and the presence of atheromatous plaques would be the technique of choice for the assessment of subclinical atherosclerosis in ACKD (Strength of Recommendation C). - C-reactive protein, measured by high-sensitivity assay (hs-CRP), is the only marker of inflammation used routinely in clinical practice. However, there is no evidence that routine determination of hs-CRP improves the outcomes of therapeutic decisions on cardiovascular protection in either the general population or ACKD population. Therefore, routine CRP determination cannot be recommended in patients with CKD to stratify their cardiovascular risk (Strength of Recommendation C).

[Animal models of peritoneal dialysis: relevance, difficulties, and future]. / Modelos animales de diálisis peritoneal: relevancia, dificultades y futuro.

The studies performed with human peritoneal biopsies of peritoneal dialysis -patients have demonstrated that exposure to peritoneal dialysis fluid induce peritoneal deterioration. The main alterations of peritoneal membrane are fibrosis and angiogenesis that ends with the failure of the ultrafiltration capacity of the peritoneal membrane. These studies are descriptivist and scarcely help to investigate the mechanisms and stages involved on the process. Therefore, it is necessary to supply the deficiencies presented by the studies with patients. The experimental models have strongly contributed to the knowledge of the pathologic process that is induced by the continuous exposition of the peritoneal membrane to the dialysis fluids. Most of the peritoneal dialysis studies use the rat as the experimental animal. Due to the difficulty of working with small animals, few studies have been done in mice. However, models in mice offers great advantages, as long as they allow us to employ different strains and genetically modified animals. We have recently developed an experimental model in mouse of exposure of the peritoneal membrane to dialysis fluids, which resembles the process of peritoneal damage that take place during peritoneal dialysis treatment in human patients.

Apoptosis: from advances in PD to therapeutic targets in DM.

A high glucose concentration is shared by peritoneal dialysis (PD) and diabetes mellitus (DM). High glucose leads to tissue injury in diabetes. Peritoneal dialysis research has emphasized the role of glucose degradation products in tissue injury. Apoptosis induction is one of the mechanisms of tissue injury induced both by glucose and glucose degradation products. We now review the role of apoptosis and its regulation by glucose degradation products in antibacterial defense and loss of renal function in diabetes mellitus and peritoneal dialysis. The pathogenic role of the recently identified glucose degradation product 3,4-di-deoxyglucosone- 3-ene (3,4-DGE) is detailed. Available therapeutic strategies include the use of peritoneal dialysis solutions containing a low concentration of glucose degradation products. Based on preclinical results, specific targeting of apoptosis regulatory factor should be explored in the clinical setting.

[Theoretical bases for the proposal of a randomized, controlled, open label clinical trial to assess the efficacy of adding bemiparin to the icodextrin solution in patients on peritoneal dialysis with peritoneal transport disorders [FRIAT-BEM-2005-01]]. / Bases teóricas para la propuesta de un ensayo clínico aleatorizado, controlado y abierto, para evaluar la eficacia de la adición de bemiparina a la solución de icodextrina en pacientes en diálisis peritoneal con trastornos del transporte peritoneal [FRIAT-BEM-2005-01].

Multiple investigations performed on peritoneal pathophysiology during peritoneal dialysis (PD) suggest that intraperitoneal heparin might modify most of the causes of membrane deterioration. The actions described favouring this idea are: 1) Peritoneal Chronic inflammation alters peritoneal function and hepraine has anti-inflammatory properties. 2) Peritoneal fibrosis related to peritoneal dialysis or traumatic injury may be avoided or limited with heparin. 3) Heparine induces tPA synthesis by mesothelial cells, which represents a potentiation of fibrinolytic action. 4) Heparine, specifically low-molecular weight heparin, inhibits angiogenesis. 5) Intraperitoneal heparin favors the removal of advanced glycosilation end products in PD. 6) Animal models and clinical studies with small series of patients have demonstrated an improvement of peritoneal function with intraperitoneal heparine use. 7) Until now, no adverse effects of the intraperitoneal heparin use have been found. In consequence, it is a plausible hypothesis to consider that intraperitoneal heparin may favourably modify peritoneal function in patients under peritoneal dialysis.

Angiotensin II activates nuclear transcription factor kappaB through AT(1) and AT(2) in vascular smooth muscle cells: molecular mechanisms.

Nuclear factor-kappaB (NF-kappaB) regulates many genes involved in vascular physiopathology. We have previously observed in vivo NF-kappaB activation in injured vessels that diminished by angiotensin-converting enzyme inhibition. In the present work, we investigated the effect of angiotensin II (Ang II) on NF-kappaB activity in rat vascular smooth muscle cells, evaluating the molecular mechanisms and the specific receptor subtype involved. Ang II increased NF-kappaB DNA binding (5-fold, 10(-)(9) mol/L at 1 hour; electrophoretic mobility shift assay), nuclear translocation of p50/p65 subunits, and cytosolic inhibitor kappaBalpha (IkappaBalpha) degradation. Ang II elicited NF-kappaB-mediated transcription (transfection of a reporter gene) and expression of NF-kappaB-related genes (monocyte chemoattractant protein-1 and angiotensinogen). AT(1) (DUP753) and AT(2) (PD123319 and CGP42112) receptor antagonists inhibited Ang II-induced NF-kappaB DNA binding in a dose-dependent manner ( approximately 85% for each one; 10(-)(5) mol/L at 1 hour). The AT(2) agonist p-aminophenylalanine(6)-Ang II augmented NF-kappaB binding (4.6-fold, 10(-)(9) mol/L at 1 hour), p65 nuclear levels, and transcription of an NF-kappaB reporter gene. AT(1) antagonist markedly inhibited NF-kappaB-mediated transcription and gene expression. Some differences between AT(1)/AT(2) intracellular signals were found. Antioxidants and ceramide inhibitors, but not protein kinase C inhibitors, diminished NF-kappaB activation elicited by both Ang II and the AT(2) agonist, while tyrosine kinase inhibitors only decreased Ang II-induced NF-kappaB activity. Our results demonstrate that Ang II activates NF-kappaB via AT(1) and AT(2), although NF-kappaB-mediated transcription occurred mainly through AT(1). Both receptors share some signaling pathways (oxygen radicals and ceramide); however, tyrosine kinases only participate in AT(1)/NF-kappaB responses. These data provide novel insights into Ang II actions, suggesting a potential implication of the AT(2) in the pathobiology of vascular cells.

[Vasoprotective effects of statins and angiotensin II blockers in atherothrombosis]. / Efectos vasoprotectores de las estatinas y los bloqueantes de la angiotensina II en la aterotrombosis.

Cardiovascular disease, including atherothrombosis, is the most frequent cause of mortality in the Western World. In the last years, major advances have been made in the pathogenesis of this disease. Currently, the drugs most widely used are the inhibitors of the HMG-CoA reductase (statins) and the antihypertensive drugs, mainly angiotensin II blockers. The first group has been shown to be effective on cardiovascular disease due to atherothrombosis, and the second group on hypertensive disease. Nevertheless, recent data suggest that these two situations can improve with the concomitant use of both drugs.

Albumin-induced apoptosis of tubular cells is modulated by BASP1.

Albuminuria promotes tubular injury and cell death, and is associated with faster progression of chronic kidney disease (CKD) to end-stage renal disease. However, the molecular mechanisms regulating tubular cell death in response to albuminuria are not fully understood. Brain abundant signal protein 1 (BASP1) was recently shown to mediate glucose-induced apoptosis in tubular cells. We have studied the role of BASP1 in albumin-induced tubular cell death. BASP1 expression was studied in experimental puromycin aminonucleoside-induced nephrotic syndrome in rats and in human nephrotic syndrome. The role of BASP1 in albumin-induced apoptosis was studied in cultured human HK2 proximal tubular epithelial cells. Puromycin aminonucleoside induced proteinuria and increased total kidney BASP1 mRNA and protein expression. Immunohistochemistry localized the increased BASP1 to tubular cells. BASP1 expression colocalized with deoxynucleotidyl-transferase-mediated dUTP nick-end labeling staining for apoptotic cells. Increased tubular BASP1 expression was observed in human proteinuric nephropathy by immunohistochemistry, providing evidence for potential clinical relevance. In cultured tubular cells, albumin induced apoptosis and increased BASP1 mRNA and protein expression at 6-48 h. Confocal microscopy localized the increased BASP1 expression in albumin-treated cells mainly to the perinuclear area. A peripheral location near the cell membrane was more conspicuous in albumin-treated apoptotic cells, where it colocalized with actin. Inhibition of BASP1 expression by a BASP1 siRNA protected from albumin-induced apoptosis. In conclusion, albumin-induced apoptosis in tubular cells is BASP1-dependent. This information may be used to design novel therapeutic approaches to slow CKD progression based on protection of tubular cells from the adverse consequences of albuminuria.

Effects and interactions of endothelin-1 and angiotensin II on matrix protein expression and synthesis and mesangial cell growth.

Mesangial cell growth and accumulation of extracellular matrix proteins constitute key features of progressive glomerular injury. Endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictor agents, evoke a number of similar responses in mesangial cells. In rat mesangial cells, we compared ET-1 and Ang II effects on matrix protein production and cell proliferation as well as the potential interaction between the two hormones. When cells in 0.5% fetal calf serum were incubated for 24 hours with various concentrations of ET-1 or Ang II, both peptides stimulated, in a dose-dependent manner, fibronectin and type IV collagen mRNA expression, fibronectin synthesis, and mitogenesis. Incubation with specific receptor antagonists of both hormones demonstrated that endothelin subtype A (ETA) and angiotensin type 1 (AT1) receptors were involved. Preincubation of cells with two different protein kinase C inhibitors or with a neutralizing anti-transforming growth factor-beta antibody, but not an unrelated IgG, diminished the peptide-induced fibronectin synthesis. A dual interrelation seems to exist between ET-1 and Ang II. Thus, the AT1 receptor antagonist losartan and the angiotensin-converting enzyme inhibitors quinaprilat and captopril diminished the ET-1-mediated effects, whereas, the ETA receptor antagonist BQ-123 diminished the Ang II-induced fibronectin synthesis and mesangial cell proliferation. Our results suggest that ET-1 and Ang II stimulate matrix protein synthesis and mesangial cell mitogenesis through ETA and AT1 receptors, respectively, by complicated mechanisms, implicating protein kinase C activation, synthesis of transforming growth factor-beta, and release of one peptide by the other. These data could be important for a better understanding of the participation of vasoactive substances in the pathogenesis of glomerulosclerosis.

Angiotensin III up-regulates genes involved in kidney damage in mesangial cells and renal interstitial fibroblasts.

Angiotensin (Ang) II is considered the effector peptide of the renin-angiotensin system (RAS) that acts as a renal growth factor. Some studies have shown that the angiotensin degradation product Ang III presents some biological activities, though its role in renal pathology has not been explored. We have observed that in renal interstitial fibroblasts Ang III induces c-fos gene expression, suggesting a potential role of Ang III in the control of cell proliferation. To study the involvement of Ang III in matrix regulation, we determined whether Ang III increased TGF-beta gene expression and fibronectin production in cultured rat mesangial cells and renal interstitial fibroblasts, the main effector cells in glomerular and interstitial fibrosis, respectively. In both cell types, treatment with Ang III (10(-7) M) for six hours up-regulated gene expression of transforming growth factor-beta 1 (TGF-beta 1; 2.3- and 2.2-fold, respectively). This peptide also increased fibronectin production in renal interstitial fibroblasts. All these data suggest that Ang III could contribute to matrix accumulation. Activation of local RAS has been described during renal damage. Renal cells express angiotensinogen mRNA that was up-regulated in response to Ang II and Ang III stimulation, and therefore both peptides may participate in the generation of angiotensin peptides in the kidney. In conclusion, our results suggest that the angiotensin degradation product Ang III could participate in the pathogenesis of key events of renal diseases, supporting the hypothesis that other peptides of the RAS besides Ang II may be involved in renal injury.

Angiotensin II: a double-edged sword in inflammation.

Clinical and basic research has increased our knowledge of the actions of the vasoactive hormone angiotensin II (Ang II), showing that it has multifunctional properties beyond its hemodynamic effects. It is commonly accepted that Ang II is a growth factor that participates in pathological settings, including renal diseases. However, a new aspect of this peptide is coming into focus: its potential role as a proinflammatory modulator. In this review, we summarize the apparently confusing information about the properties of Ang II and discuss its relations to the inflammatory process, as well as the potential mechanisms mediated by activation of nuclear transcription factors. Ang II seems to participate in the key events of the inflammatory response: First, it increases vascular permeability (via prostaglandins and vascular endothelial cell growth factor/vascular permeability factor) thus initiating the inflammatory process. Second, it participates in the recruitment of infiltrating cells into the tissues through direct activation of the inflammatory cells or by regulation of the expression of adhesion molecules and chemokines by resident cells. Finally, Ang II may contribute to tissue repair by regulation of cell growth and matrix synthesis. The renin-angiotensin system (RAS) thus appears to serve as a vascular inflammatory regulator and may even participate in immunologically-induced inflammation. However, more often activation of the RAS has an undesirable outcome, such as overhealing, because the inflammatory repair response itself involves a functionally imperfect system.

Involvement of angiotensin II and endothelin in matrix protein production and renal sclerosis.

PURPOSE: To review the evidence that links angiotensin II and endothelin as growth factors and as modifiers of extracellular matrix synthesis in renal cells, with particular reference to the effects of angiotensin converting enzyme (ACE) inhibition in models of renal injury. IN VITRO STUDIES: In cultured mesangial cells, both angiotensin II and endothelin promote contraction, proliferation/hypertrophy, signal transduction pathways, the activation of early growth genes, and the generation of inflammatory mediators, cytokines and growth factors. Both hormones have been shown to promote the synthesis of fibronectin and collagen in a dose-dependent manner. ACE inhibition attenuates the effect of endothelin-1, one of three isoforms of endothelin. ANIMAL STUDIES: In experimental models of renal injury, chiefly in those characterized by increased intraglomerular pressure, ACE inhibition has reduced proteinuria and glomerular and interstitial sclerosis. HUMAN STUDIES: ACE inhibition has been shown to have major beneficial effects in patients with diabetic nephropathy, even in those with normal blood pressure. CONCLUSIONS: Although the renal-protective effects of ACE inhibitors in experimental and human renal injury may reflect systemic and/or local hemodynamic effects of these drugs, their modulatory actions on extracellular matrix synthesis and proteinuria may contribute to the benefit of ACE-inhibitor therapy.

Comparación del tiempo de cicatrización entre N-butil cianoacrilato y ácido poliglicólico en caninos sometidos a esterilización / Comparison of healing times between N-butyl cyanoacrylate and polyglycolic acid in canines subjected to sterilization

RESUMEN El objetivo de este estudio fue comparar el tiempo de cicatrización entre N-butil cianocrilato (NBC) y ácido poliglicólico (AC) sobre el cierre tisular en caninos sometidos a operaciones de esterilización. Se realizó un estudio observacional, comparativo y descriptivo sobre una población de 80 perros (Canis familiaris) vivos. Los animales se dividieron en cuatro grupos de 20 perros cada uno según sexo y material de sutura, así: primero grupo, hembras que recibieron NBC; segundo grupo, machos y NBC; tercero grupo, hembras y AC como medio de sutura en piel y cuarto grupo, machos y AC. La técnica quirúrgica en hembras fue oforosalpingohisterectomia y en machos orquiectomia. El tiempo de cierre fue estadísticamente (p < 0,05) menor en los grupos de machos y hembras con el adhesivo NBC comparado con los dos grupos tratados con AC. Se concluye que el NBC es un material seguro y eficaz como medio de fijación tisular en ambas técnicas quirúrgicas que disminuye los días de cierre y recuperación.

ABSTRACT The aim of this study was to compare N-butyl cyanoacrylate (NBC) and polyglycolic acid (AC) in tissue closure in canines subjected to sterilization operations. An observational, comparative and descriptive study was conducted in a population of 80 living dogs (Canis familiaris). The animals were divided into 4 groups of 20 dogs each, according to gender and suture material: the first group consisted of females that received NBC; the second group consisted of males that received NBC; the third group consisted of females that received AC as skin suture; and the fourth group was formed by males that received AC. The surgical technique in females was salpingo-oophorectomy, and in males it was orchiectomy. The closure time was statistically (p < 0.05) lower in the male and female groups with the NBC adhesive in comparison to the two groups treated with AC. It was concluded that NBC is a safe and effective material for tissue fixation in both surgical techniques, decreasing times of closure and recovery.