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The rapid development of augmented reality (AR) applications has led to wide adoption in education, acting as a supporting tool to increase the transmitted information. However, children need to improve their knowledge of plants, their categories, their conceptual framework as well as the human relationship with them. This research studies the learning outcomes of a plant-themed augmented reality mobile application and its key features designed for 1st graders of primary school. The results of the research showed that there was no statistically significant improvement between the experimental group and the control group.

Background Hyperuricemia is common among kidney transplant recipients and has been associated with worse graft outcome. Since episodes of acute cellular rejection and chronic humoral rejection contribute to decreased graft survival, in this study the effect of uric acid on cellular and humoral alloimmunity was evaluated. Methods Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method in which humoral alloimmunity was induced in one-way MLR. Then the de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MRLs were used against resting PBMC similar to the stimulator cells of the above MLRs. Results Uric acid at a concentration above its crystallization threshold increased cellular proliferation in two-way MLRs. Supernatants from one-way MLRs performed in the presence of uric acid were more cytotoxic against PBMC from individuals that had conferred the stimulator cells for the above MLRs. Conclusions Uric acid increases both cellular and humoral alloimmunity in human PBMC. These results offer a possible pathogenetic mechanism for the observed relation between hyperuricemia and worse kidney allograft survival.

Uric acid is a known danger associated molecular pattern molecule able to induce inflammation following internalization of its crystals by cells of the innate immune system. By activating antigen-presenting cells, urate boosts adaptive immunity as well. Furthermore, urate crystals can induce proliferation of isolated T-cells, which are unable to phagocytose crystal particles. In light of the evidence that urate crystals can also activate dendritic cells and macrophages without prior internalization but through sequestration of lipid rafts (and consequently receptors clustering in a non specific manner), the authors evaluated whether such a mechanism is involved in the direct activation of T-cells by urate crystals. In the present study, isolated human T-cells were cultured with or without urate at a concentration above its crystallization level. The expression and phosphorylation state of the T-cell receptor (TCR) complex zeta chain and the expression of the master regulator of cell proliferation c-Myc were assessed by western blotting. T-cell proliferation was measured by bromodeoxyuridine assay. Collectively, the results indicated that urate increased zeta chain phosphorylation indicating that it induces activation of TCR complex directly, since zeta chain phosphorylation takes place at the cell membrane and is a very proximal event in TCR complex-mediated signal transduction. In parallel, urate increased the expression of the transcription factor c-Myc and induced T-cell proliferation. In conclusion, urate crystals directly activate the TCR complex and induce T-cell proliferation.

Along with infections, ultrafiltration failure due to the toxicity of glucose-containing peritoneal dialysis (PD) solutions is the Achilles&rsquo, heel of PD method. Triggered by the protective effect of general control nonderepressible-2 (GCN-2) kinase activation against high-glucose conditions in other cell types, we evaluated whether the same occurs in human peritoneal mesothelial cells. We activated GCN-2 kinase with halofuginone or tryptophanol, and assessed the impact of this intervention on glucose transporter-1, glucose transporter-3, and sodium-glucose cotransporter-1, glucose influx, reactive oxygen species (ROS), and the events that result in glucotoxicity. These involve the inhibition of glyceraldehyde 3-phosphate dehydrogenase and the diversion of upstream glycolytic products to the aldose pathway (assessed by D-sorbitol), the lipid synthesis pathway (assessed by protein kinase C activity), the hexosamine pathway (determined by O-linked &beta, N-acetyl glucosamine-modified proteins), and the advanced glycation end products generation pathway (assessed by methylglyoxal). Then, we examined the production of the profibrotic transforming growth factor-&beta, 1 (TGF-&beta, 1), the pro-inflammatory interleukin-8 (IL-8). Cell apoptosis was assessed by cleaved caspase-3, and mesothelial to mesenchymal transition (MMT) was evaluated by &alpha, smooth muscle actin protein. High-glucose conditions increased glucose transporters, glucose influx, ROS, all the high-glucose-induced harmful pathways, TGF-&beta, 1 and IL-8, cell apoptosis, and MMT. Halofuginone and tryptophanol inhibited all of the above high glucose-induced alterations, indicating that activation of GCN-2 kinase ameliorates glucotoxicity in human peritoneal mesothelial cells, preserves their integrity, and prevents MMT. Whether such a strategy could be applied in the clinic to avoid ultrafiltration failure in PD patients remains to be investigated.

Chronic antibody-mediated rejection remains a major cause of late graft loss. Regarding cellular alloimmunity, the immunosuppressive properties of indoleamine 2,3-dioxygenase (IDO) have been well investigated; however, little is known of its effects on humoral alloimmunity. Therefore, the present study aimed to evaluate the effects of IDO on humoral alloimmunity. We developed a method for the induction of humoral alloimmunity in a one-way mixed lymphocyte reaction (MLR), which was measured with an antibody-mediated complement-dependent cytotoxicity assay using resting cells, which are similar to the stimulator cells of the aforementioned MLR. In parallel, cellular alloimmunity was assessed in two-way MLRs. The IDO inhibitor 1-methyl-DL-tryptophan was used for evaluating the role of IDO. In order to investigate whether the pathways known to serve a role in the effects of IDO on T cells are applied in humoral alloimmunity, the general control nonderepressible-2 (GCN-2) kinase activator tryptophanol and the aryl hydrocarbon receptor (AhR) inhibitor CH223191 were employed. The IDO inhibitor was revealed to increased cellular autoimmunity, but was decreased by the GCN-2 kinase activator. Unexpectedly, the AhR inhibitor decreased cellular alloimmunity. In addition, the IDO inhibitor was observed to suppress humoral alloimmunity, which may occur in manners independent of GCN-2 kinase AhR. The present study proposed that IDO may decrease humoral alloimmunity in primary human peripheral blood mononuclear cells via pathways that differ to those associated with its effect on T cells.

BackgroundUrate in its crystal form is a known danger-associated molecular pattern, which after its internalization activates cells of the innate immune system. However, by inducing lipid raft sequestration and clustering of membrane-bound proteins with immunoreceptor tyrosine-based activation motifs, urate crystals can also activate cells of the innate immune system without previous internalization. Also, urate crystals trigger T-cell receptor signal transduction and induce T-cell proliferation. In this study, we evaluated whether urate crystals can also initiate B-cell receptor (BCR) signal transduction and promote B-cell proliferation.MethodsB cells were isolated from the blood of 10 individuals and cultured with or without urate at a concentration of 10 mg/dL, at which crystallization occurs. Phosphorylated Igα (CD79A) and c-Myc were assessed by Western blotting and B-cell proliferation with BrdU assay.ResultsUrate increased the level of phosphorylated Igα, a component of the BCR complex. Phosphorylation of Igα is the very proximal event in BCR signal transduction. Also, urate increased the expression of c-Myc, an essential transcription factor for BCR-induced B-cell proliferation. Finally, urate induces B-cell proliferation.ConclusionsUrate crystals trigger BCR signal transduction and induce B-cell proliferation. The clinical significance of urate-induced B-cell activation remains to be elucidated.

Considering the referred beneficial effects of protein restriction on diabetic nephropathy (DN) and the role of renal endothelium in its pathogenesis, we evaluated the effect of general control nonderepressible 2 (GCN2) kinase activation, a sensor of amino acid deprivation, on known detrimental molecular pathways in primary human glomerular endothelial cells (GEnC). GEnC were cultured under normal or high-glucose conditions in the presence or not of the GCN2 kinase activator, tryptophanol. Glucose transporter 1 (GLUT1) expression was assessed by western blotting and reactive oxygen species (ROS) using a fluorogenic probe. Activities of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and protein kinase C (PKC) were assessed by commercial activity assays, sorbitol colorimetrically, methylglyoxal by ELISA and O-linked β-N-acetyl glucosamine (O-GlcNAc)-modified proteins by western blotting. High glucose induced GLUT1 expression, increased ROS and inhibited GAPDH. Also it increased the polyol pathway product sorbitol, PKC activity, the level of the O-GlcNAc-modified proteins that produced by the hexosamine pathway and the advanced glycation endproducts’ precursor methylglyoxal. Co-treatment of GEnC with tryptophanol restored the above high-glucose-induced alterations. Activation of GCN2 kinase protects GEnC from high-glucose-induced harmful molecular pathways. By inhibiting concurrently many pathways involved in DN pathogenesis, GCN2 kinase may serve as a pharmaceutical target for the treatment of DN.

Amino acid deprivation activates general control nonderepressible 2 (GCN2) kinase and inhibits mammalian target of rapamycin (mTOR), affecting the immune response. In this study, the effects of GCN2 kinase activation or mTOR inhibition on human alloreactive CD4+ T-cells were evaluated. The mixed lymphocyte reaction, as a model of alloreactivity, the GCN2 kinase activator, tryptophanol (TRP), and the mTOR complex 1 inhibitor, rapamycin (RAP), were used. Both TRP and RAP suppressed cell proliferation and induced cell apoptosis. These events were p53-independent in the case of RAP, but were accompanied by an increase in p53 levels in the case of TRP. TRP decreased the levels of the Th2 signature transcription factor, GATA-3, as RAP did, yet the latter also decreased the levels of the Th1 and Th17 signature transcription factors, T-bet and RORγt, whereas it increased the levels of the Treg signature transcription factor, FoxP3. Accordingly, TRP decreased the production of interleukin (IL)-4, as RAP did, but RAP also decreased the levels of interferon-γ (IFN-γ) and IL-17. Both TRP and RAP increased the levels of IL-10. As regards hypoxia-inducible factor-1α (HIF-1α), which upregulates the Th17/Treg ratio, its levels were decreased by RAP. TRP increased the HIF-1α levels, which however, remained inactive. In conclusion, our findings indicate that, in primary human alloreactive CD4+ T-cells, the two systems that sense amino acid deprivation affect cell proliferation, apoptosis and differentiation in different ways or through different mechanisms. Both mTOR inhibition and GCN2 kinase activation exert immunosuppressive effects, since they inhibit cell proliferation and induce apoptosis. As regards CD4+ T-cell differentiation, mTOR inhibition exerted a more profound effect, since it suppressed differentiation into the Th1, Th2 and Th17 lineages, while it induced Treg differentiation. On the contrary, the activation of GCN2 kinase suppressed only Th2 differentiation.

Comparative biology may reveal novel therapeutic strategies against human diseases. Ischemia‑reperfusion (IR) injury induces a number of diseases. It is known that hibernating mammals survive IR since during hibernation, prolonged periods of torpor with a marked decrease in blood flow and breathing rate are interrupted by short periods of arousal. In the present study, the differences in the character‑ istics of endoplasmic reticulum (ER) stress and the subsequent unfolded protein response, which are induced by IR and may cause cell death among humans, mice or the native hibernator Syrian hamster were examined in vitro using renal proximal tubular epithelial cells (RPTECs) derived from these three sources. RPTECs were subjected to anoxia or reoxygenation, both at 37˚C. Cell death was measured by LDH release assay. ER stress was assessed by determining the levels of phos‑ phorylated protein kinase RNA‑like ER kinase, ubiquitinated proteins and Bcl‑2‑associated X protein (Bax) by western blot analysis. For proteasomal activity, a specific assay was used. The results revealed that anoxia induced ER stress in all the evaluated RPTECs, from which only the hamster‑derived RPTECs recovered during reoxygenation. Anoxia and reoxy‑ genation increased protein ubiquitination in the human‑ and mouse‑derived RPTECs, whereas this was decreased in the hamster‑derived RPTECs. Anoxia enhanced proteasomal activity in all the evaluated RPTECs. In the human‑ and mouse‑derived RPTECs, reoxygenation reduced proteasomal activity, which remained high in the hamster‑derived RPTECs. Anoxia and reoxygenation increased Bax expression and induced cell death in the human‑ and mouse‑derived RPTECs, while neither Bax overexpression nor cell death occurred in the hamster‑derived RPTECs. Thus, on the whole, the find‑ ings of this study demonstrate that compared to human‑ or mouse‑derived RPTECs, those derived from the hamster recover more rapidly from ER stress following warm anoxia‑reoxygenation, possibly due to increased proteasomal function.

Ischemia-reperfusion (I-R) injury causes several diseases, including acute kidney injury. Hibernating mammals survive periods of torpor with a marked drop in tissue perfusion, interspersed with periods of arousal, and consequently I-R injury. In the present study, sensitivity to anoxia and/or reoxygenation and alterations in cellular ATP and homeostasis of the two most energy consuming processes, protein translation and Na+-K+-ATPase function, were evaluated in renal proximal tubular epithelial cells of mouse or native hibernator hamster origin. Compared with the mouse cells, the hamster cells were less sensitive to anoxia and reoxygenation and ATP was preserved under anoxia. Anoxia triggered mechanisms that suppress protein translation in both species. However, under anoxia, the activity of ATPase, which is mostly attributed to Na+-K+-ATPase function, remained stable in the hamster cells but decreased in the mouse cells. In normoxia, ATPase activity in hamster cells was considerably lower than that in mouse cells. As the Na+-K+-ATPase pump preserves the ion gradient against passive leakage through ion channels, the lower energy demand for the function of this pump in hamster cells may indicate less ion leakage due to fewer ion channels. In accordance with this hypothesis, ouabain-treated hamster cells had a higher survival rate than mouse cells, indicating fewer ion channels and consequently slower deregulation of intracellular ion concentration and cell death due to Na+-K+-ATPase inhibition. Therefore, it is likely that the conserved energy from the suppression of protein translation is adequate enough to support the lower energy demand for Na+-K+-ATPase function and cell survival of hamster cells under anoxia. Clarifying how cells of a native hibernator manage energy under warm I-R may reveal novel and possible clinically applicable pathways for preventing I-R injury.

Activated T cells rely on aerobic glycolysis and glutaminolysis in order to proliferate and differentiate into effector cells. Therefore, intervention in these metabolic pathways inhibits proliferation. The aim of the present study was to evaluate the effects of Krebs' cycle inhibition at the level of malate dehydrogenase-2 (MDH2) in human activated T cells using the MDH2 inhibitor LW6. Activated T cells from healthy volunteers were cultured in the presence or absence of LW6 and cytotoxicity, cell proliferation and the expression levels of hypoxia-inducible factor (HIF)-1α, c-Myc, p53, cleaved caspase-3 and certain enzymes involved in glucose metabolism and glutaminolysis were evaluated. The results revealed that LW6 was not toxic and decreased apoptosis and the levels of the pro-apoptotic tumor suppressor p53. In addition, LW6 inhibited T-cell proliferation and decreased the levels of c-Myc, HIF-1α, glucose transporter-1, hexokinase-II, lactate dehydrogenase-A and phosphorylated pyruvate dehydrogenase. By contrast, LW6 increased the levels of pyruvate dehydrogenase. These alterations may lead to decreased production of pyruvate, which preferentially enters into the Krebs' cycle. Furthermore, LW6 decreased the levels of glutaminase-2, while increasing those of glutaminase-1, which may preserve glutaminolysis, and possibly pyruvate-malate cycling, potentially protecting the cells from energy collapse. In summary, the inhibition of MDH2 in activated T cells abrogates proliferation without adversely affecting cell survival. Adaptations of cellular glucose and glutamine metabolism may prevent energy collapse.

Summary Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and exerts immunosuppressive effects on CD4+ T cells. One mechanism is through the inhibition of aerobic glycolysis. Another prerequisite for T-cell proliferation and differentiation into effector cells is increased fatty acid (FA) synthesis. The effect of IDO on enzymes involved in FA synthesis was evaluated in primary human cells both in mixed lymphocyte reactions in the presence or not of the IDO inhibitor 1-dl-methyl-tryptophan, and in stimulated CD4+ T cells in the presence or not of the general control non-derepressible 2 (GCN2) kinase activator tryptophanol (TRP). IDO or TRP inhibited cell proliferation. By assessing the level of GCN2 kinase or mammalian target of rapamycin complex 1 substrates along with a kynurenine free system we showed that IDO exerts its effect mainly through activation of GCN2 kinase. IDO or TRP down-regulated ATP-citrate lyase and acetyl coenzyme A carboxylase 1, key enzymes involved in FA synthesis. Also, IDO or TRP altered the expression of enzymes that control the availability of carbon atoms for FA synthesis, such as lactate dehydrogenase-A, pyruvate dehydrogenase, glutaminase 1 and glutaminase 2, in a way that inhibits FA synthesis. In conclusion, IDO through GCN2 kinase activation inhibits CD4+ T-cell proliferation and down-regulates key enzymes that directly or indirectly promote FA synthesis, a prerequisite for CD4+ T-cell proliferation and differentiation into effector cell lineages.

Mitochondrial reactive oxygen species (ROS) overproduction in capillary endothelial cells is a prerequisite for the development of diabetic nephropathy. Inhibition of xanthine oxidase, another ROS generator, ameliorates experimental diabetic nephropathy. To test the hypothesis that the initial high glucose-induced ROS production by the mitochondria activates xanthine oxidase, which afterward remains as the major source of ROS, we cultured primary human glomerular endothelial cells (GEnC) under normal or high-glucose conditions, with or without the xanthine oxidase inhibitor allopurinol. ROS generation and nitric oxide synthase (NOS) activity were assessed by chemiluminescence or colorimetrically. Levels of intercellular adhesion molecule 1 (ICAM-1), p53 and phosphorylated p53 (p-p53) were assessed by western blotting. Allopurinol prevented high glucose-induced ROS generation indicating that xanthine oxidase is the major source of ROS. Allopurinol protected GEnC from endothelial dysfunction since it prevented the high glucose-induced decrease in NOS activity and increase in ICAM-1 expression. Allopurinol reduced p53 and p-p53 levels induced by high glucose suggesting an axis of xanthine oxidase-derived ROS, DNA damage, p53 stabilization and endothelial dysfunction that may contribute to the pathogenesis of diabetic nephropathy. Allopurinol protects GEnC from high glucose-induced ROS generation, p53 overexpression and endothelial dysfunction. These data provide a pathogenetic mechanism that supports the results of experimental and clinical studies about the beneficial effect of xanthine oxidase inhibitors on the development of diabetic nephropathy.

Acute cellular rejection is the major cause of immune-mediated graft failure early in the course of kidney transplantation, whereas chronic antibody-mediated rejection is a major contributor to graft loss in the late post-transplant phase. Based mainly on the results of short-term studies, the calcineurin inhibitor tacrolimus prevails over the mammalian target of rapamycin (mTOR) inhibitors. However, the toxicity profile of the two drug categories differs, making the interchange between them appealing. In this study, the effect of tacrolimus and of the mTOR inhibitor everolimus on cellular and humoral alloimmunity was evaluated. Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity, we developed a method in which humoral alloimmunity was induced in a one-way MLR. The de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MLRs were used against resting PBMC similar to the stimulator cells of the forementioned MLRs. Tacrolimus and everolimus were used at concentrations near their upper recommended trough levels. In two-way MLRs, tacrolimus inhibited cell proliferation more than everolimus. In one-way MLRs, tacrolimus and everolimus decreased alloantibody production to the same extent. In human cell cultures, everolimus is inferior to tacrolimus in inhibiting cellular alloimmunity, but equally effective as regards humoral alloimmunity. Thus, everolimus might be a safe alternative in case of tacrolimus toxicity, particularly after the early period of kidney transplantation.

Hyperuricaemia is common among kidney transplant recipients and has been associated with worse graft outcome. Since episodes of acute cellular rejection and chronic humoral rejection contribute to decreased graft survival, in this study the effect of uric acid on cellular and humoral alloimmunity was evaluated.Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method in which humoral alloimmunity was induced in one-way MLR. Then the de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MRLs were used against resting PBMC similar to the stimulator cells of the above MLRs.Uric acid at a concentration above its crystallization threshold increased cellular proliferation in two-way MLRs. Supernatants from one-way MLRs performed in the presence of uric acid were more cytotoxic against PBMC from individuals that had conferred the stimulator cells for the above MLRs.Uric acid increases both cellular and humoral alloimmunity in human PBMC. These results offer a possible pathogenetic mechanism for the observed relation between hyperuricaemia and worse kidney allograft survival.

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity by inhibiting T-cell proliferation and altering glucose metabolism. The tumor suppressor p53 also alters these cellular processes with similar results. The effect of IDO on p53 and on glucose metabolism was evaluated in alloreactive T cells. Mixed-lymphocyte reactions (MLRs) were performed in the presence or not of the IDO inhibitor, 1-dl-methyl-tryptophan (1-MT) and/or the p53 inhibitor, pifithrin-α (PFT). Cell proliferation, glucose consumption and lactate production were assessed. 1-MT increased cell proliferation, glucose influx and lactate production, whereas PFT enhanced cell proliferation and glucose influx, leaving lactate production unaffected. In MLR-derived T cells, protein analysis revealed that IDO activated general control non-derepressible 2 kinase and induced p53, p-p53 (p53 phosphorylated at serine 15) and p21. In addition, both IDO and p53 decreased glucose transporter 1 and TP53-induced glycolysis and apoptosis regulator and increased synthesis of cytochrome c oxidase 2. IDO also reduced lactate dehydrogenase-A and glutaminase 2 levels, whereas p53 left them unaffected. Neither 1-MT nor PFT affected glucose-6-phosphate dehydrogenase. In conclusion, in alloreactive T cells, IDO increases p53 levels, and both IDO and p53 inhibit cell proliferation, glucose consumption and glycolysis. Lactate production and glutaminolysis are also suppressed by IDO, but not by p53.

Disturbed iron homeostasis contributes to resistance to recombinant human erythropoietin (rHuEpo) in hemodialysis (HD) patients. Although increased hepcidin, which downregulates the iron exporter ferroportin, had been incriminated, such an association has not been confirmed. Albeit not universally accepted, it has been supported that in case of copper deficiency, decreased activity of multicopper oxidases induces endocytosis and degradation of ferroportin. Ferroportin in monocytes, serum copper, ceruloplasmin and markers of iron status were measured, and associations with rHuEpo resistance index (ERI) were evaluated. After a 4-week washout period from iron treatment, 34 HD patients and 20 healthy volunteers enrolled in the study. Ferroportin was assessed by means of Western blotting, copper colorimetrically, whereas ceruloplasmin with enzyme-linked immunosorbent assay. Hemoglobin, serum iron, ferritin and transferrin saturation (TSAT) were also measured. Ferroportin in monocytes of HD patients was decreased. Serum copper, ceruloplasmin, iron and TSAT were decreased. No correlation between copper or ceruloplasmin and ferroportin was detected. ERI was negatively correlated with ferroportin and all the markers of iron adequacy, but not with copper or ceruloplasmin. Although copper deficiency and decreased ferroportin are common in HD patients, copper might not play role in ferroportin level in monocytes and in iron metabolism in this population.

A 77-year-old man, 11 years under chronic hemodialysis treatment for chronic renal failure of unknown origin, presented with anterior chest pain, dyspnea with paradoxical breathing, and sternal instability after a simple fall from a standing height. Patient underwent three-vessel coronary artery bypass grafting 31 months ago. Computed tomography with three-dimensional volume rendering showed sternal nonunion with a great gap between the two halves of the sternum and at least one fracture in the left half of the sternum. A successful surgical repair followed. Patient suffered from severe secondary hyperparathyroidism for many years. Despite treatment with sevelamer, paricalcitol and cinacalcet, intact parathyroid hormone was 1682 pg/mL. During the last 5 years, serum intact parathyroid hormone remained steadily above 1000 pg/mL. Patient refused parathyroidectomy in the past. We assume that long-lasting severe hyperparathyroidism contributed to this rare and life-threatening complication of median sternotomy in our patient, due to the detrimental effect of hyperparathyroidism on bone metabolism and its association with increased incidence of bone fractures and defect in bone fracture healing.

We describe a case of a patient with a functional kidney transplant who was admitted to our department with clinically evident central vein stenosis (CVS) 7 years after the removal of a central venous catheter (CVC) from the right internal jugular vein. The catheter was used as a hemodialysis access for a 2-month period. In the interval before his last admission, the patient suffered two episodes of deep vein thrombosis. Investigation revealed heterozygosity for factor V Leiden, the most common inherited thrombophilia encountered in 5% of Caucasians, and anticoagulation treatment was started. Magnetic resonance angiography showed stenosis just after the convergence of the right subclavian vein with the internal jugular vein to the innominate vein. Transluminal angioplasty restored venous patency and right upper arm edema resolved. Coexistence of CVS, accompanied by hemodynamic changes and endothelial dysfunction, with thrombophilia fulfill all the elements of the Virchow's triad. Therefore, the patient was at great risk for central vein thrombosis, from which he was possibly protected by the early administration of anticoagulant treatment. This case indicates that CVS can be asymptomatic for several years after CVC removal and also raises the question if thrombophilia workup and investigation for CVS may be beneficial in every patient with CVC placement in order to avoid any harmful outcomes.

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-methyl-dl-tryptophan (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

Studies detected an association between visfatin and markers of iron metabolism in patients with insulin resistance. In this study, such a relation was evaluated in hemodialysis (HD) patients. Also relations between visfatin and hepcidin, demands for recombinant human erythropoietin (rHuEpo), inflammation, and situations characterized by insulin resistance were evaluated.After a four-week washout period from iron treatment, 33 HD patients and 20 healthy volunteers enrolled in the study. Serum visfatin, hepcidin, and interleukin-6 (IL-6) were assessed by means of enzyme-linked immunosorbent assay. Hemoglobin, serum iron, ferritin, and transferrin saturation (TSAT) were also measured.Visfatin was markedly increased in HD patients. Visfatin levels did not differ between diabetics and non-diabetics. No relation was detected between visfatin and body mass index or IL-6 in HD patients. From the markers of iron metabolism, the hepcidin included, visfatin was related only to TSAT. A strong positive relation was revealed between visfatin and hemoglobin, whereas visfatin was inversely related to rHuEpo dose. Resistance to rHuEpo index was inversely and independently of TSAT related to visfatin.Visfatin is increased in HD patients and it is associated with decreased demands for rHuEpo.

Proteasome generates peptides for presentation to CD8+ T-cells by antigen presenting cells (APCs) and peripheral cells. Inflammation alters proteasome to immunoproteasome, producing a different pool of peptides. The effect of immunoproteasome expression in APCs and in peripheral cells on CD8+ T-cell self-tolerance was evaluated. Splenocytes (SP) were used as a source of APCs and CD8+ T-cells. Renal epithelial cells (RC) from the same mouse strain were used as peripheral cells. Lipopolysaccharide (LPS) was used for increasing immunoproteasome expression, whereas transduction with lentiviral particles encoding short-hairpin RNA targeting LMP7 (LnLMP7) were used for decreasing the expression of the LMP7 subunit of the immunoproteasome in SP and/or RC. LMP7 expression was tested with Western blotting, while the cytotoxicity of isolated CD8+ T-cells against RC with LDH release assay. LPS increased LMP7 expression in SP, while did not affect its expression in RC. LnLMP7 decreased LMP7 expression in both LPS-treated and untreated SP and RC. CD8+ T-cell auto-reactivity increased in case of LPS-treated APCs, further enhanced in the event of both APCs and RC treated with LPS. Transduction of APCs or RC with LnLMP7 decreased CD8+ T-cell auto-reactivity, which was further decreased in case of concurrent transduction of both APCs and RC. In conclusion, in controlled in vitro conditions, exposure to LPS of APCs and peripheral cells induces CD8+ T-cell auto-reactivity. Over-expression of the LMP7 subunit of the immunoproteasome in APCs due to LPS exposure, as well as LMP7 expression in peripheral cells, are required for CD8+ T-cell auto-reactivity.

Diabetic nephropathy is the leading cause of end-stage renal disease. Diabetes mellitus is characterized by generalized endothelial dysfunction. However, recent data also emphasizes the role of local renal endothelium dysfunction in the pathogenesis of diabetic nephropathy. Hyperglycemia triggers a complex network of signal-transduction molecules, transcription factors, and mediators that culminate in endothelial dysfunction. In the glomerulus, vascular endothelial growth factor-A (VEGF)-induced neoangiogenesis may contribute to the initial hyperfiltration and microalbuminuria due to increased filtration area and immaturity of the neovessels, respectively. However, subsequent decrease in podocytes number decreases VEGF production resulting in capillary rarefaction and decreased glomerular filtration rate (GFR). Decreased nitric oxide availability also plays a significant role in the development of advanced lesions of diabetic nephropathy through disruption of glomerular autoregulation, uncontrolled VEGF action, release of prothrombotic substances by endothelial cells and angiotensin-II-independent aldosterone production. In addition, disturbances in endothelial glycocalyx contribute to decreased permselectivity and microalbuminuria; whereas there are recent evidences that reduced glomerular fenestral endothelium leads to decreased GFR levels. Endothelial repair mechanisms are also impaired in diabetes, since circulating endothelial progenitor cells number is decreased in diabetic patients with microalbuminuria. Finally, in the context of elevated profibrotic cytokine transforming growth factor-β levels, endothelial cells also confer to the deteriorating process of fibrosis in advanced diabetic nephropathy through endothelial to mesenchymal transition.

Under growth conditions, angiogenin is translocated into the nucleus, where it enhances ribosomal RNA transcription, facilitating increased protein synthesis and cellular proliferation. During stress conditions, angiogenin is sequestered in the cytoplasm, where it cleaves transfer RNA (tRNA) to produce tRNA-derived, stress-induced small RNAs (tiRNAs) that inhibit global protein synthesis, but increase the translation of anti-apoptotic factors. In the present study, the role of angiogenin in the human alloreactive immune response was evaluated using mixed lymphocyte reactions (MLRs) and neamine, an inhibitor of angiogenin nuclear translocation. In MLRs, angiogenin production was significantly (P

In atherosclerosis-associated pathologic entities characterized by malnutrition and inflammation, L-tryptophan (TRP) levels are low. Insulin resistance is an independent cardiovascular risk factor and induces endothelial dysfunction by increasing fatty acid oxidation. It is also associated with inflammation and low TRP levels. Low TRP levels have been related to worse cardiovascular outcome. This study evaluated the effect of TRP depletion on endothelial dysfunction under conditions that imitate insulin resistance. Fatty acid oxidation, harmful pathways due to increased fatty acid oxidation, and endothelial dysfunction were assessed in primary human aortic endothelial cells cultured under normal glucose, low insulin conditions in the presence or absence of TRP. TRP depletion activated general control non-derepressible 2 kinase and inhibited aryl hydrocarbon receptor. It increased fatty acid oxidation by increasing expression and activity of carnitine palmitoyltransferase 1. Elevated fatty acid oxidation increased the formation of reactive oxygen species (ROS) triggering the polyol and hexosamine pathways, and enhancing protein kinase C activity and methylglyoxal production. TRP absence inhibited nitric oxide synthase activity in a ROS-dependent way, whereas it increased the expression of ICAM-1 and VCAM-1 in a ROS independent and possibly p53-dependent manner. Thus, TRP depletion, an amino acid whose low levels have been related to worse cardiovascular outcome and to inflammatory atherosclerosis-associated pathologic entities, under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through ROS-dependent and independent pathways. These findings may offer new insights at the molecular mechanisms involved in accelerated atherosclerosis that frequently accompanies malnutrition and inflammation.

Hemodialysis (HD) patients are characterized by impaired T-cell function at least in part because of T-cell zeta-chain downregulation due to inflammation. Angiogenin responds as an acute phase reactant, is increased in HD patients, suppresses T-cell function and increased angiogenin level is co-localized with T-cell zeta-chain downregulation in various pathologies. Angiogenin can inhibit translation of proteins, which lack internal ribosomal entry sites in the corresponding mRNAs. In this study, the possible effect of angiogenin on T-cell zeta-chain downregulation was evaluated. Thirty HD patients and 21 healthy volunteers participated. T-cell zeta-chain expression was assessed with flow cytometry, plasma angiogenin and serum IL-6 with ELISA and serum C-reactive protein with an immunoturbidimetric method. Two available software tools were used for predicting the presence or not of internal ribosomal entry sites in T-cell zeta-chain mRNA sequence. In silico analysis of T-cell zeta-chain mRNA sequence failed to reveal the presence of internal ribosomal entry sites. T-cell zeta-chain expression was lower in HD patients than in healthy volunteers (1.86 ± 0.63 vs. 4.73 ± 3.22). In HD patients, C-reactive protein as well as IL-6 were higher than in healthy volunteers (10.04 ± 15.13 mg/L vs. 3.43 ± 0.98 mg/L and 15.06 ± 13.08 pg/mL vs. 2.11 ± 2.10 pg/mL respectively). Angiogenin was higher in HD patients than in healthy volunteers (483.20 ± 154.07 ng/mL vs. 259.98 ± 64.15 ng/mL). Neither C-reactive protein, nor IL-6 was associated with angiogenin or T-cell zeta-chain expression. Angiogenin concentration was negatively related to the expression of T-cell zeta-chain (r = -0.410, P = 0.025). Increased angiogenin may contribute to decreased T-cell zeta-chain expression in HD patients.

Acquired immunity is impaired in hemodialysis (HD) patients, and decreased T-cell number may contribute. Indoleamine 2,3-dioxygenase (IDO) and arginase type I (ARG) catabolize tryptophane and arginine, respectively, and exert proapoptotic and antiproliferative effects on T-cells. Plasma levels of IDO and ARG and their relation to blood T-cell number were evaluated in HD patients.Thirty-two HD patients and 20 healthy controls participated in the study. Plasma IDO and ARG were measured by means of enzyme-linked immunosorbent assay. T-cell number was assessed by means of flow cytometry.IDO concentration was significantly higher in HD patients than in healthy volunteers (44.30 ± 31.83 ng/mL vs. 21.28 ± 26.21 ng/mL, p = 0.009). There was a trend for higher ARG concentration in HD patients (13.43 ± 11.91 ng/mL) than in healthy volunteers (9.56 ± 4.03 ng/mL), which, however, did not reach statistic significance (p = 0.099). Absolute T-cell count was significantly lower in HD patients than in healthy controls (1176.99 ± 567.71 cells/mm3 vs. 1519.85 ± 594.96 cells/mm3, p = 0.040). Absolute blood T-cell number was inversely related to plasma IDO (r = -0.490, p = 0.004) and to plasma ARG (r = -0.387, p = 0.029) concentrations.Plasma IDO and ARG may contribute to decreased blood T-cell count in HD patients.

Perilipin-1 surrounds lipid droplets in both adipocytes and in atheroma plaque foam cells and controls access of lipases to the lipid core. In hemodialysis (HD) patients, dyslipidemia, malnutrition, inflammation and atherosclerosis are common. Thirty-six HD patients and 28 healthy volunteers were enrolled into the study. Ten HD patients suffered from coronary heart disease (CHD). Perilipin-1, triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), body mass index, albumin, geriatric nutritional risk index, normalized protein catabolic rate, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured. Perilipin-1 did not differ between HD patients and healthy volunteers. IL-6 and TNF-α were higher in HD patients. The evaluated nutritional markers and the markers of inflammation did not differ between HD patients with high perilipin-1 levels and HD patients with low perilipin-1 levels. Regarding the lipid profile, only HDL-C differed between HD patients with high perilipin-1 levels and HD patients with low perilipin-1 levels, and it was higher in the first subgroup. Perilipin-1 was significantly higher in HD patients without CHD. Perilipin-1 is detectable in the serum of HD patients and it is associated with increased HDL-C and decreased incidence of CHD.

Ischemia&ndash, reperfusion injury contributes to the pathogenesis of many diseases, with acute kidney injury included. Hibernating mammals survive prolonged bouts of deep torpor with a dramatic drop in blood pressure, heart, and breathing rates, interspersed with short periods of arousal and, consequently, ischemia&ndash, reperfusion injury. Clarifying the differences under warm anoxia or reoxygenation between human cells and cells from a native hibernator may reveal interventions for rendering human cells resistant to ischemia&ndash, reperfusion injury. Human and hamster renal proximal tubular epithelial cells (RPTECs) were cultured under warm anoxia or reoxygenation. Mouse RPTECs were used as a phylogenetic control for hamster cells. Cell death was assessed by both cell imaging and lactate dehydrogenase (LDH) release assay, apoptosis by cleaved caspase-3, autophagy by microtubule-associated protein 1-light chain 3 B II (LC3B-II) to LC3B-I ratio, necroptosis by phosphorylated mixed-lineage kinase domain-like pseudokinase, reactive oxygen species (ROS) fluorometrically, and lipid peroxidation, the end-point of ferroptosis, by malondialdehyde. Human cells died after short periods of warm anoxia or reoxygenation, whereas hamster cells were extremely resistant. In human cells, apoptosis contributed to cell death under both anoxia and reoxygenation. Although under reoxygenation, ROS increased in both human and hamster RPTECs, lipid peroxidation-induced cell death was detected only in human cells. Autophagy was observed only in human cells under both conditions. Necroptosis was not detected in any of the evaluated cells. Clarifying the ways that are responsible for hamster RPTECs escaping from apoptosis and lipid peroxidation-induced cell death may reveal interventions for preventing ischemia&ndash, reperfusion-induced acute kidney injury in humans.

Cardiovascular disease, and mainly coronary heart disease (CHD), is the leading cause of death in hemodialysis (HD) patients. Non-traditional risk factors may play an important role in this population. Arginase is known to contribute directly to atherosclerosis progression and to counteract the beneficial effects of nitric oxide. HD could be considered as an inflammatory condition. Inflammation contributes to atherosclerosis progression and influences both arginase and nitric oxide synthase expression. In the present study, serum arginase type I was evaluated as a marker of CHD in HD patients. The markers of inflammation interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were also assessed. Sixty-eight HD patients and 24 healthy volunteers were enrolled into the study. Twenty HD patients suffered from CHD confirmed with coronary angiography, while the remaining 48 HD patients were asymptomatic. Serum arginase type I, IL-6 and TNF-α were measured with ELISA. During 24 months follow-up, none of the asymptomatic subjects developed symptoms of CHD. IL-6 and TNF-α levels were increased in HD patients, but did not differ between HD patients with or without CHD. On the contrary, arginase levels did not differ between healthy subjects and HD patients, but were twice higher in HD patients with CHD than in HD patients without CHD (22.41 ± 15.47 ng/ml vs. 10.16 ± 8.13 ng/ml). Arginase type I may contribute to the pathogenesis of CHD in HD patients and its serum levels could be used as a marker of CHD in this population.

Vitamin D and its analogues proved to exert immunomodulatory effects. Paricalcitol is a vitamin D analogue that is safe. It has been used for years in the treatment of secondary hyperparathyroidism in hemodialysis patients and, importantly, it is less calcemic than vitamin D. In this study the immunomodulatory/anti-inflammatory properties of paricalcitol were evaluated in vitro.Ten healthy volunteers enrolled into the study. Peripheral blood mononuclear cells (PBMC) at a concentration of 10(6) cells per well were cultured for 48 h in the presence or not of lipopolysaccharide (LPS) (100 ng/ml) and in the presence or not of paricalcitol (10(-8) M). TNF-alpha and IL-8 were measured in the supernatants by ELISA.Basal TNF-alpha concentration (50.3 +/- 22 pg/ml) was reduced by paricalcitol (44.1 +/- 23.2 pg/ml). LPS increased TNF-alpha concentration (150.0 +/- 81.7 pg/ml), but paricalcitol reduced it (121.1 +/- 69.0 pg/ml). The effect of paricalcitol on IL-8 production was more profound. Basal IL-8 concentration (1926 +/- 455 pg/ml) was reduced by paricalcitol (1273 +/- 472 pg/ml). LPS increased IL-8 concentration (2361 +/- 385 pg/ml), but paricalcitol returned it to its basal level (1849 +/- 417 pg/ml).The in vitro inhibition of transforming growth factor alpha and interleukin 8 by paricalcitol confirms the immunomodulatory properties of this vitamin D analogue.

Anemia is a common complication in hemodialysis (HD) patients. Despite the great success of recombinant human erythropoietin in clinical practice, resistance to this therapy is common. Additionally, nephrologists frequently witness a rapid and significant drop in their patients' hematocrit during the course of various acute events that regularly take place in this sensitive population. Hepcidin, a recently identified peptide, may mediate this development in many instances. Hepcidin production is regulated by hypoxia/anemia, iron status, and importantly, inflammation. This peptide can block iron absorption by the duodenum, iron release from both the liver (the main iron storage pool) and, more significantly, the macrophages interrupting iron recycling between senescent red cells and the reticuloendothelial system. The decreased availability of iron for erythropoiesis leads to the anemia of chronic disease or, in HD patients, aggravate an already existing anemia HD is now widely considered an inflammatory state probably accounting for the increased serum hepcidin levels that have been associated with it. The physiology of hepcidin and its possible contribution to the pathogenesis of anemia in HD patients are the subject of this review.

Oxidative stress is increased in hemodialysis (HD) patients and contributes to the increased morbidity and mortality in this population. Vitamin E is an antioxidant agent. In the present study the effect of prolonged oral alpha-tocopherol administration on the antioxidant defense system was evaluated. The antioxidant factors plasma total antioxidant status (TAS), red blood cell superoxide dismutase (SOD) activity and glutathione peroxidase (GPX) activity were evaluated with spectrometry in 27 HD patients. Measurements were performed before and after oral administration of alpha-tocopherol at a dose of 500 mg/d for a one-year period. Twenty HD patients received a placebo and 22 healthy volunteers served as controls. TAS was increased in HD patients. No difference was detected in SOD and GPX activity between HD patients and healthy volunteers. Tocopherol administration induced a significant decrease in TAS and SOD activity. Levels of GPX activity remained unaffected. All the evaluated factors remained stable in the HD patients receiving a placebo. Prolonged oral alpha-tocopherol administration in HD patients induces a decrease in some components of the antioxidant defense system, raising the possibility for a pro-oxidative role of vitamin E. Vitamin E is an antioxidant agent, but it is also known to have pro-oxidant action under special conditions that can be encountered in HD patients.

Renal osteodystrophy is very common in hemodialysis (HD) patients. HD is a chronic inflammatory state. Studies in other pathological entities have shown an impact of chronic inflammation on bone metabolism. In the present study, the impact of chronic inflammation on bone turnover in HD patients was evaluated.Thirty-three anuric HD patients free of other pathological conditions or medications that affect immune system or bone metabolism and 30 healthy volunteers enrolled into the study. Intact parathyroid hormone (iPTH), the markers of inflammation IL-6 and CRP, as well as the markers of bone turnover osteocalcin (OCN) and beta-isomerized C-terminal cross-linked peptide of collagen type I (beta-CTx) were measured in the serum.All evaluated factors were increased in HD patients. In the HD group, the serum marker of osteoblastic activity OCN was related inversely to patients' age (r = -0.469, p = 0.006), CRP (rho = -0.460, p = 0.007), and IL-6 (r = -0.485, p = 0.004) but positively to iPTH (r = 0.707, p0.001). Similarly, the serum marker of osteoclastic activity beta-CTx was related inversely to patients' age (r = -0.383, p = -0.028), CRP (rho = -0.466, p = 0.006), and IL-6 (r = -0.460, p = 0.007) but positively to iPTH (r = 0.657, p0.001). Multiple linear regression analysis revealed that IL-6 affects bone turnover independently of PTH and to the opposite direction.Chronic inflammation has a negative impact on bone turnover in HD patients. Certainly, further research and large clinical trials are needed for definite conclusions and for clarifying the exact molecular mechanisms implicated in the interaction between the immune system and bone metabolism in HD patients.

Background: Clinical and experimental data indicate a deficient immune response in hemodialysis (HD) patients. ζ-Chain phosphorylation is an early and central event in the process that follows antigen recognition by the T cell antigen receptor (TCR). T cell ζ-chain is downregulated in many chronic inflammatory states, such as cancer, autoimmune disease and chronic infection. HD is also characterized as a chronic inflammatory state. The aim of the present study was to evaluate T cell ζ-chain expression in HD patients. Patients and Methods: Thirty-three stable HD patients and 30 healthy volunteers were enrolled into the study. T cell count, the percentage of ζ-chain-positive T cells, as well as T cell ζ-chain mean fluorescence intensity (MFI) were evaluated with flow cytometry. The inflammatory markers C-reactive protein, interleukin-6 and tumor necrosis factor-α were measured in the serum by means of ELISA. Results: All the evaluated markers of inflammation were increased in HD patients. In these patients, T cell ζ-chain MFI was decreased. CD3-Ε MFI did not differ between the two groups indicating that among the TCR complex constituents, ζ-chain is selectively downregulated. Conclusions: HD is a state of chronic inflammation. Like in other pathological chronic inflammatory conditions, T cell ζ-chain is downregulated in HD patients. Since ζ-chain plays a key role in the transduction of the signal that follows antigen recognition by the TCR, its downregulation could be responsible for the deficient cellular immune response observed in HD patients.

Acquired immunity disturbances in hemodialysis (HD) patients are many and diverse. They are caused by uremia per se, the HD procedure, chronic renal failure complications, and therapeutic interventions for their treatment. Current data suggest that acquired immunity disturbances in HD patients concern mainly the T-lymphocyte and the antigen-presenting cell (APC). The T-lymphocyte-dependent immune response is deficient, predisposing to infections and inadequate response to vaccinations. In addition, APCs are preactivated, which seems to be responsible for the malnutrition-inflammation-atherosclerosis syndrome, and also affects T-lymphocyte function. At the molecular level it is assumed that the interaction between the APC and the T-lymphocyte is impaired. This disturbance is likely to concern the signal that results from the interaction between the major histocompatibility complex:peptide complex on APC surfaces and T-cell receptors on T-lymphocyte surfaces, or the signal that results from the interaction among the co-receptors of these two cells. The aim of the present review was to collect and classify the available clinical and experimental data in this area. Although many pieces are still missing from the puzzle, a better understanding of the responsible molecular mechanisms, will potentially lead to increased survival and a better quality of life in HD patients.

Cardiovascular disease is the leading cause of death in hemodialysis (HD) patients. Coronary artery calcification (CAC) is considered a marker of atherosclerosis and coronary artery disease (CAD). The CAC progression and factors that influence it were evaluated during a 30-month period.Forty HD patients without a history of CAD were enrolled into the study. CAC score was assessed with conventional CT repeated every six months. The circulating factors of phosphorous, calcium, calcium-phosphorous product, intact parathyroid hormone, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, lipoprotein-alpha, albumin, high sensitivity C-reactive protein, and fibrinogen were measured monthly. Hypertension and calcium intake during the study period were taken into account as well.At baseline, CAC score was correlated with age and duration of HD therapy. From all evaluated factors, CAC initiation was influenced only by older age and C-reactive protein. CAC, when it was started, was aggravated continuously and was influenced only by elevated serum phosphorous and calcium-phosphorous product. Hypertension, lipid profile, and calcium intake did not affect CAC initiation or progression.Once CAC progression starts, it is an uninterrupted process. The roles of inflammation and abnormal calcium-phosphorous metabolism in CAC differ. Inflammation is the major factor that contributes in CAC initiation. Elevated serum phosphorous and calcium-phosphorous product accelerates CAC progression.

Ischemic acute kidney injury is characterized by apoptosis of tubular epithelial cells. Proteasome plays a key role in cellular processes such as proliferation, apoptosis and inflammation. The results of animal studies about the effect of proteasome inhibitors on the course of ischemic acute kidney injury are controversial. Primary human renal tubular epithelial cells were cultured with or without the hypoxia mimetic CoCl2 and with or without the proteasome inhibitor CEP-18770 and/or the immunoproteasome inhibitor ONX-0914. The level of the proteasome subunit β5, the immunoproteasome subunits LMP7 and LMP2, the function of these proteolytic machines, HIF-1α and its transcriptional target lactate dehydrogenase-A, p53 and its transcriptional targets TP53-inducible glycolysis and apoptosis regulator and p21, and finally of activated cleaved caspase-3 were assessed by means of western blotting. CoCl2 decreased the expression of β5, LMP7 and LMP2, as well as the activity of proteasome and immunoproteasome. It increased HIF-1α and its function, along with p53 and its function and induced apoptosis. CEP-18770 and ONX-0914 induced the above alterations toward the same directions as CoCl2 does. In CoCl2-treated cells, pretreatment with CEP-18770 and/or ONX-0914 potentiates the changes induced by CoCl2 alone. CoCl2, CEP-18770 and ONX-0914 induce apoptosis in human renal tubular epithelial cells. Importantly, proteasome or immunoproteasome inhibitors are rather toxic than beneficial in human renal tubular epithelial cells treated with the hypoxia mimetic CoCl2.

Hemodialysis patients suffer from susceptibility to infections. Inflammation upregulates indoleamine 2,3-dioxygenase (IDO) in the antigen-presenting cells, which suppresses T-cell function. Plasma IDO activity or protein expression is increased in hemodialysis patients and is associated with immune disturbances. This observation, however, does not consider many factors, importantly the source of IDO, which has to be the antigen-presenting cells in order IDO to exert its immunosuppressive effect in the microenvironment of the immune response. In this study, monocytes were isolated from 30 hemodialysis patients and 20 healthy volunteers and IDO was assessed by Western blotting. The IDO level in the monocytes of hemodialysis patients was significantly, almost 3-fold, higher than in the monocytes of healthy volunteers. This localization enables IDO to exert its immunosuppressive effect and supports conclusions of previous studies that used more indirect methods for assessing the role of this enzyme in the context of the immune response in hemodialysis patients.

Although kidney transplantation is the best therapy for end-stage renal disease, rejection remains a concern, and currently available immunosuppressive agents contribute to morbidity and mortality. Thus, novel immunosuppressive drugs are required. Dichloroacetate (DCA) is already used in the treatment of congenital lactic acidosis and characterized by limited toxicity. As DCA inhibits aerobic glycolysis, which is a prerequisite for CD4+ T-cell proliferation and differentiation into effector T-cells, its possible immunosuppressive role in mixed lymphocyte reaction (MLR), a model of alloreactivity, was investigated. Glucose and lactate concentrations were measured in the supernatants, and cell proliferation was assessed immunoenzymatically. CD4+ T‑cells were then isolated from the MLRs and the expression of cleaved caspase‑3, various enzymes involved in glycolysis, and the signature transcription factors of CD4+ T‑cell subsets were evaluated by western blotting. In MLRs, DCA decreased glucose consumption and aerobic glycolysis, while it exerted a negligible effect on cell proliferation. In CD4+ T‑cells, DCA induced apoptosis, and decreased the expression of glucose trasporter‑1, hexokinase II, lactate dehydrogenase‑A and phosphorylated pyruvate dehydrogenase, while it increased total pyruvate dehydrogenase. In addition, DCA increased the expression of transcription factor forkhead box P3, whereas it decreased the expression of T‑box transcription factor TBX21, trans‑acting T-cell-specific transcription factor GATA‑3 and retinoic acid receptor related orphan receptor‑γt. In conclusion, in alloreactive CD4+ T‑cells, DCA inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T‑cell subset. These characteristics render it a promising immunosuppressive agent in the field of transplantation.

Indoleamine 2,3‑dioxygenase (IDO), through L‑tryptophan depletion, activates general control non‑derepressible (GCN) 2 kinase and suppresses T‑cell proliferation, in addition to suppressing aerobic glycolysis and glutaminolysis, which are required for these rapidly proliferating cells. A number of, however not all of these alterations, are partially mediated through IDO‑induced p53 upregulation. In two‑way mixed lymphocyte reactions (MLRs), IDO reduced cellular proliferation. In MLR‑derived T‑cells, IDO induced the expression levels of p53 and p21, however concurrently reduced the levels of ζ‑chain, c‑Myc, lactate dehydrogenase A (LDH‑A) and glutaminase (GLS)2. However, p53 had no effect on the expression of the above proteins. These results were recapitulated in T‑cells activated with anti‑CD2, anti‑CD3 and anti‑CD28 by direct activation of the GCN2 kinase with tryptophanol. In conclusion, IDO, through GCN2 kinase activation, downregulates the levels of TCR‑complex ζ‑chain and c‑Myc, resulting in the suppression of T‑cell proliferation and a reduction in the levels of LDH‑A and GLS2, which are key enzymes involved in aerobic glycolysis and glutaminolysis, respectively.

Introduction: Prohepcidin is the precursor of hepcidin, a liver-derived peptide involved in iron metabolism by blocking its intestinal absorption and its release by the reticuloendothelial system. Iron overload and inflammation increase hepcidin expression, whereas anemia and hypoxia suppress it. In the present study prohepcidin levels were determined in the serum of hemodialysis (HD) patients and its correlations with iron metabolism markers, C-reactive protein (CRP) and hematocrit (Hct) were assessed. Patients and Methods: Forty-sixHD patients and 22 healthy volunteers were enrolled in the study. Hct, serum prohepcidin, CRP, iron, ferritin, transferrin saturation and transferrin receptors were measured. The weekly erythropoietin dose, last-month intravenous iron dose and the patients’ demographics were recorded. Results: In comparison to the healthy volunteers, the HD patients had higher serum ferritin, transferrin receptors and CRP, lower serum iron and similar transferrin saturation and prohepcidin levels. In the patient group prohepcidin levels were negatively correlated with Hct but not with any other of the examined parameters. Multiple linear regression analysis considering age, inflammation, iron adequacy, erythropoietin dose and prohepcidin levels revealed that prohepcidin was the predominant determinant of Hct. Conclusions: Taking into account the low Hct levels in the HD patients of our study, it seems plausible that the prohepcidin levels assessed in this group are inappropriately high. These functionally high prohepcidin levels may be associated with the factors that inhibit erythropoiesis in HD patients. On the other hand, the absence of other expected correlations indicates that further studies are needed in order to definitely clarify this aspect.

The risk for tuberculosis (TB) reactivation in hemodialysis (HD) patients is increased, and screening for latent TB is recommended. The tuberculin skin test (TST) is inaccurate because of the high incidence of anergy in skin testing in HD patients and its inability to differentiate latent from eradicated TB infection. In this study, use of serum antilipoarabinomannan (anti-LAM) antibody detection for the diagnosis of latent TB in a dialysis population was evaluated.Seventy-four HD patients never treated for TB composed the first group. Forty-eight healthy volunteers never treated for TB served as controls. Twenty-one TST-positive renal transplant candidates on dialysis therapy who had completed preventive anti-TB treatment formed a third group. The TST (using the Mantoux method) and anti-LAM test (using an immunochromatographic assay) were performed in all subjects participating in the study.In the first group, a strong association was detected between results of the TST and anti-LAM test (P0.0001, chi-square test). A similar association was found in the second group. In the group of HD patients treated for TB, a positive TST result and negative anti-LAM test result was a constant finding.The association between the TST and anti-LAM test in HD patients suggests that the anti-LAM test could be a useful tool for diagnosing latent TB. The absence of anti-LAM antibodies in anti-TB-treated HD patients suggests that immune response to lipoarabinomannan lacks long-lasting memory. A negative anti-LAM test result might reflect the absence of latent TB, even in patients with a positive TST result.

Background. Anticardiolipin antibodies (ACA) have been related to an increased incidence of thrombotic episodes and atherosclerosis progression. ACA levels are elevated in hemodialysis (HD) patients. Atheroembolic episodes are the major cause of morbidity and mortality in this population. Oxidative stress has been implicated in ACA formation, and it is increased in HD patients. Vitamin E is a known antioxidant factor. In this study, the effects of prolonged oral α-tocopherol administration on ACA levels were evaluated. Methods. Serum anticardiolipin IgG antibodies (ACA-IgG) and IgM antibodies (ACA-IgM) levels were evaluated in 27 stable HD patients and 22 healthy volunteers. Then measurements were performed in the patients' group after oral administration of α-tocopherol at a dose of 500 mg/d for a 1-year period. ACA levels were assessed by solid-phase enzyme immunoassay. Results. ACA-IgG levels were higher in HD patients compared with control (13.3 ± 6.64 GPL/mL vs. 7.727 ± 18.305 GPL/mL, p < .001). This...

We evaluated the value of coronary artery calcification (CAC) score in coronary artery disease (CAD) detection in asymptomatic hemodialysis (HD) patients by evaluating the association among CAC score, exercise electrocardiography (EECG), and Thallium-201 dipyridamole scintigraphy. Correlation between aortic pulse wave velocity (PWV) and CAC score was also evaluated.CAC score was assessed with conventional computed tomography in 40 patients. Thirty patients completed EECG and 25; those with a positive CAC score and/or a positive EECG performed Thallium dipyridamole scintigraphy. Carotid-femoral PWV was assessed in all patients.There was no association among CAC score and EECG or Thallium dipyridamole scintigraphy. In contrast, CAC score was correlated with aortic PWV.The previous results question the role of CAC score in the detection of CAD in asymptomatic HD patients. The correlation between CAC score and aortic PWV raises the possibility that CAC score represents more an indicator of coronary artery medial wall calcification than a marker of CAD.