Joanna Bober

Conjugated linoleic acid isomers may diminish human macrophages adhesion to endothelial surface

Dysfunction of endothelial cells and activation of monocytes in the vascular wall are important pathogenetic factors of atherosclerosis. Conjugated linoleic acids (CLAs) can modulate the function of immune system in humans: reduce the concentration of atherogenic lipoproteins, and the intensity of inflammatory processes in the plasma. In this paper, we focus on macrophages surface integrins (β1 integrin CD49d/CD29-(VLA4); Mac-1 as well as endothelial human vein endothelial cell (HUVEC) surface adhesins: vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1)) expression in relation to CLA isomer used during cell culture. Both CLA isomers decreased expression of VLA-4 and Mac-1 on macrophages compared with control cells (cultured with bovine serum albumine (BSA) or oxidized form of low-density lipoproteins). cis-9, trans-11 CLA isomer reduced ICAM-1 and VCAM-1 expression on the endothelium surface. Strong tendency to reduce of adhesion of macrophages to HUVEC in the cells cultured with CLA isomers was observed. The potential role of cis-9, trans-11 CLA in the reduction of adhesion of macrophages to the HUVEC – one of the important steps in the inflammatory process, can be considerate. These mechanisms may contribute to the potent anti-atherosclerotic effects of CLA in vivo.

Does Glucose in Dialysis Fluid Protect Erythrocytes in Patients with Chronic Renal Failure

Background/Aims: The aim of this study was to assess the multifaceted influence of glucose present in dialyzing fluid on erythrocytes of patients with chronic renal failure (CRF) undergoing regular hemodialysis. Methods: A group of 44 subjects with CRF undergoing regular hemodialysis was studied. Two tests were used: osmotic fragility and resistance to the hemolytic agent saponin. The total content of isoprostane 8-iso-prostaglandin F2α type III (8-iPF2α-III) in plasma and erythrocyte’s membrane were determined by the ELISA method. Results: The presence of glucose in the dialysate is associated with lower intravascular hemolysis markers and high total 8-iPF2α-III concentrations in plasma. Conclusion: The presence of glucose in dialyzing fluid could protect erythrocytes. It limits hemolysis in patients with CRF, but, on the other hand, increases the oxidative processes. This kind of treatment along with other therapeutic intervention such as administration of antioxidants (e.g. α-tocopherol, ascorbic acid, N-acetylcysteine) could improve the condition of erythrocytes and outcome in CRF.

Metformina – mechanizmy działania i zastosowanie w terapii cukrzycy typu 2[i][/i]

Metformin is widely used for the treatment of type 2 diabetes mellitus. Although this biguanide derivative has been used for more than 50 years, its mechanism of action has not been fully elucidated. In this article we describe the latest achievements concerning the mechanisms of antihyperglycemic action of metformin. They include: decrease of glucose absorption in the small intestine, increase of glucose transport into cells, decrease in the plasma free fatty acid concentrations and inhibition of gluconeogenesis. Activation of AMP-activated protein kinase (AMPK) plays an important role in these processes. The latest discoveries have revealed mechanisms of anti-atherosclerotic, hypotensive and anticancer action of metformin and its impact on vein endothelial function. The pleiotropic actions of metformin include impact on plasma lipid profile, decrease of oxidative stress, and increase in plasma fibrinolytic activity. Although metformin is not metabolized, the latest research has shown that it is actively transported into hepatocytes and renal tubular epithelium, by OCT1 (organic cation transporter 1, encoded by the SLC22A1 gene) and OCT2 (organic cation transporter 2, encoded by the SLC22A2 gene), respectively. However, MATE1 transporter (multidrug and toxin extrusion 1 protein) is encoded by the SLC47A1 gene and facilitates metformin excretion from these cells into bile and urine. Metformin transporter gene polymorphisms may contribute to significant variation in drug response. Further studies of mechanisms of metformin action could contribute to its wider use for the prevention of type 2 diabetes mellitus, cancer, and Alzheimer’s disease, and for the treatment of type 1 diabetes mellitus, and polycystic ovary syndrome (PCOS).

Does Glucose Present in the Dialysate Limit Oxidative Stress in Patients Undergoing Regular Hemodialysis

Background: Decreased glucose concentration in the blood causes the inhibition of the hexose monophosphate (HMP) cycle in the erythrocyte. NADPH, which is the source of the reductive equivalents necessary for the reproduction of glutathione (GSH), is not regenerated. The presence of glucose in dialysate should provide the stability of its concentration in the blood of patients undergoing hemodialysis (HD). The aim of the study was to assess the influence of glucose in the dialysate on the intensity of oxidative stress in patients undergoing regular HD. Methods: The study comprised 43 patients hemodialyzed with dialysate containing (HD-g(+)) or not containing glucose (HD-g(–)). The concentrations of the products of reaction with thiobarbituric acid-reactive substance (TBARS) and GSH as well as the activity of erythrocyte superoxide dismutase were determined. Glucose concentrations in the blood before and immediately after dialysis were also measured. Results: After flow-through dialysis the glucose concentration in the blood decreases both when dialysate does not contain glucose (4.8 vs. 1.6 mmol/l) and when dialysate contains glucose (6.6 vs. 5.8 mmol/l). HD caused changes in the TBARS concentration: in the HD-g(+) group the concentration decreased after HD, whereas in the HD-g(–) group it increased. In both groups of patients studied the GSH concentration changed after HD; in the HD-g(–) group it decreased and in the HD-g(+) group it increased. The results obtained in the groups of patients examined were confirmed by in vitro studies. Conclusions: The presence of glucose in the dialysate guarantees the normal activity of the HMP cycle, which provides the production of reductive equivalents for the regeneration of reduced GSH – free radicals scavenger – and therefore the limitation of oxidative stress.

Influence of angiotensin I‐converting enzyme polymorphism on development of post‐transplant erythrocytosis in renal graft recipients

Abstract:  Background:  Post‐transplant erythrocytosis (PTE) is estimated at 5–20%. Angiotensin II generated by angiotensin I‐converting enzyme (ACE) stimulates erythropoiesis. The highest activity of ACE is observed in DD genotype. The question arises if ACE gene polymorphism influences PTE.

Urinary Metalloproteinases-9 and -2 and Their Inhibitors TIMP-1 and TIMP-2 are Markers of Early and Long-Term Graft Function After Renal Transplantation.

Background/Aims: Renal ischemia-reperfusion (I-R) injury (IRI) is an inseparable feature of organ transplantation and may have a negative impact on the graft, its function and survival. Acute tubular necrosis, which is reversible thanks to the regenerative capacity of renal tubular epithelial cells, is the main cause of acute renal failure secondary to IRI. MMP-2 and MMP-9 are proteolytic enzymes involved in digesting proteins that are components of the extracellular matrix (ECM) and the basement membrane of the nephrons. This way post-reperfusion MMP activation allows the inflammatory process to spread. Methods: In our studies, we focused on identifying whether the concentrations of MMP-2 and MMP-9 and their natural inhibitors TIMP-1 and TIMP-2 in urine sample at day 1 and day 30 as well as after 12 months following renal transplantation are markers of early and long-term renal function during meanly five-years observation. Moreover, in urine sampled at months 6 and 12 after renal transplantation, we determined the content of TGF-β as a graft fibrosis indicator. Results: MMP-9 concentration in the early post-transplant period is a major marker of early and long-term function of the transplanted kidney. Its increased concentration was correlated with lesions related to tubular atrophy and fibrosis in renal biopsies performed at months 3 and 12 after transplantation. Its concentration is correlated with TGF-β content in a later period. Conclusions: TIMP-1 and-2 are primarily markers of an early function of the transplanted kidney. Early post-transplant concentration of MMP-2 is a marker of proteinuria in early and long-term post-transplant periods.

Oxidative stress modulates the organization of erythrocyte membrane cytoskeleton.

BACKGROUND Apart from their main role in transporting oxygen and carbon dioxide, erythrocytes play also an important role in organism antioxidative defence. Direct exposure to reactive oxygen species (ROS) results in shortening of their half-life, even by 50%. The presence of glucose, being the substrate in pentose phosphate pathway (PPP) cycle, is one of the factors that can have influence on the level of oxidative stress. The activity of PPP increases during oxidative stress. Glucose guarantees normal PPP functioning with the production of reductive equivalents in the amounts necessary to reproduction of glutathione--nonenzymatic free radical scavenger. In available literature there are no reports regarding the changes in protein contents of erythrocyte cytoskeleton exposed to t-butyl hydroperoxide in relation to glucose presence in incubation medium. MATERIAL/METHODS Erythrocytes taken from 10 healthy subjects were used to assess the influence of generated free radicals on erythrocyte proteins and chosen parameters of oxidative stress. Erythrocytes were incubated in the solutions containing deferent concentrations of t-butyl hydroperoxide and glucose. Electrophoresis was performed on polyacrylamide gel in denaturating conditions. The contents of tryptophan in membranes was evaluated spectrofluorometrically. RESULTS/CONCLUSIONS In vitro conditions oxidative stress leads to protein damage in erythrocyte cytoskeleton, both in proteins inside the cell as well as having contact with extracellular environment. In consequence, the amount of low-molecular proteins--mainly globin, which bind to cytoskeleton, increases. This process takes place independently of glucose presence in incubation medium. One of the element of protein cytoskeleton, tryptophan, also undergoes degradation. The decrease of its contents is higher during erythrocyte exposure to t-BOOH in environment containing glucose, what can suggest prooxidative influence of glucose in conditions in vitro.

Oxidative Stress Indices in Rats Under Immunosuppression

Immunosuppressants lead to generation of reactive oxygen species (ROS). Oxidative stress (OxS) can initiate chronic allograft nephropathy (CAN). The most active antioxidant enzymes, superoxide dysmutase (SOD) and catalase (CAT), are present in erythrocytes. Glutathione peroxidase (GPx) is produced in the proximal tubules of nephrons. Malonyldialdehyde (MDA) concentrations are a marker of OxS intensity in plasma. In vitro and animal model studies have shown increased or decreased OxS during treatment with tacrolimus (Tac) or cyclosporine (CyA). Results obtained in humans after solid organ transplantation have been contradictory, because of confounding factors such as ischemia-reperfusion injury, donor and recipient ages, endothelial injury, and comorbidity. The aim of this study was to assess the intensity of OxS among rats under chronic immunosuppression (IS) without a transplantation. We examined 49 male Wistar rats. IS started at 12 weeks of age was continued for 6 months: group I were controls (n=7); group II, Tac+sirolimus (Rapamycin [Rapa])+corticosteroids (CS; n=6); group III, CyA+Rapa+CS (n=4 of which 2 died); group IV, Rapa+mycophenolate mofetil (MMF)+CS (n=6); group V, CyA+MMF+CS (n=6); group VI, CsA+MMF+CS for 3 months followed by conversion to Rapa (n=6); group VII, Tac+MMF+CS (n=6 rats); and group VIII, Tac+MMF+CS for 3 months followed by conversion to Rapa (n=6). The drug doses were as follows: Tac 4 mg/kg/d; MMF 20 mg/kg/d; CyA 5mg/kg/d; Rapa 0.5 mg/kg/d; and CS 4 mg/kg/d. Multiple regression analysis revealed that all IS drugs decreased GPx activity (P<.001) except CS, which increased it (P<.0001). Multiple regression analysis showed that CsA and Tac decreased plasma MDA concentrations (P<.01), whereas CS increased them (P<.05). In conclusion, all IS drugs except CS damage proximal tubules of nephrons.

Effect of Hemodialysis on the Content of Fatty Acids in Monolayers of Erythrocyte Membranes in Patients with Chronic Renal Failure

Lipid metabolism disorders are found in patients with chronic renal failure (CRF). Changes in the content of fatty acids of the phospholipid fraction of erythrocyte membranes can lead to changes in the rheological properties. The objective of our study was to assess the effect of hemodialysis on the composition of fatty acids in two fractions of phospholipids: sphingomyelin (SPH, representative of the external monolayer) and phosphatidylethanolamine (PE, representative of the internal monolayer). Venous blood was drawn from patient with CRF before and after the HD procedure. Lipids from the erythrocyte stroma were extracted using the Rose and Oklander method and then were separated into phospholipid fractions using thin-layer chromatography (TLC). PE and SPH fractions were extracted, and the fatty acid profile was determined using gas chromatography (Perkin Elmer 8400; RTx 2330 column; length: 105 m). In the phospholipid fractions tested, a high content of saturated FA with a medium carbon chain (C 16:0 to C 18:2) and a long carbon chain such as C 24:0, C 24:1; C 22:6; and C 26:0 was found. The HD procedure affected the FA profile in the fractions tested. The proportion of saturated and unsaturated long-chain FA (above 18 C) increased in PE. However, the content of medium-chain FA C 16:0 to C 18:1 decreased. A significant decrease in the content of the majority of long-chain FA could be noted in SPH. The ratio of unsaturated (U) to saturated (S) fatty acids in the SPH fraction increased. Hemodialysis has a significant effect on the content of fatty acids in the PE and SPH fractions of erythrocyte membranes in patients with CRF.

Influence of Glucose in Dialyzing Fluid on Purine Concentrations in Hemodialyzed Patients with Chronic Renal Failure

Background: In chronic renal failure the accumulation of some purine nucleotides (in erythrocytes) develops both in patients undergoing conservative treatment and in hemodialyzed patients. The aim of the study was: (1) To find if hemodialysis (HD) sessions using dialyzing fluid containing glucose leads to an increase in ATP concentration and changes in the concentration of other nucleotides, nucleosides and oxypurines in erythrocytes. The potential consequence of such purine concentration changes is the increase of 2,3-DPG concentration and an improved transportation of oxygen in erythrocytes which are more resistant to hemolysis. (2) To compare blood concentrations of purine nucleotides, nucleosides and oxypurines in patients undergoing chronic HD with dialyzing fluid containing or lacking glucose. Significant differences could suggest the long-term influence of glucose in dialyzing fluid on erythrocyte energetic state. Methods: Whole blood nucleotide concentrations were evaluated with the use of a high-performance liquid chromatography technique. Results: Before the HD session the patients in the ‘plus glucose’ group had significantly higher concentrations of ATP, ADP, AMP, TAN, NAD, NADP, GTP + GDP, GMP, Urd and HYP than patients in the ‘no glucose’ group. After the HD the patients in the ‘plus glucose’ group had significantly higher concentrations of ADP, AMP, TAN, NAD, NADP, Urd and HYP than in the ‘no glucose’ group. Both before and after the HD session, the uric acid concentrations and AEC were significantly lower in the ‘plus glucose’ group than in the ‘no glucose’ group. A significant decrease in the whole blood hypoxanthine (p < 0.05) and uric acid (p < 0.001) concentrations after HD was found in the ‘no glucose’ group while a significant increase in ADP concentration (p < 0.05) was detected in the patients’ erythrocytes in the ‘plus glucose’ group. In this group a significant decrease of GTP + GDP and GMP (p < 0.05), uric acid concentration (p < 0.001) and adenylate energy charge (p < 0.05) were observed after the dialysis. However, no significant differences in nucleotide concentrations before and after the HD were found in the ‘no glucose’ group. Conclusion: The presence of glucose in the dialyzing fluid causes a significant modification of the energetic state of cells which is reflected by the purines’ and their metabolites’ concentrations in the erythrocytes. Higher ATP concentrations in patients with renal failure who have been dialyzed with the fluid containing glucose can be considered as an organism adaptation to a decreased amount of RBC and hemoglobin concentration.