Search

Objective To evaluate the effect of advanced glycation end-products (AGEs) and the inhibitor of their formation, aminoguanidine, on tumor necrosis factor-α (TNFα) production (as a functional marker) by rat peritoneal macrophages (PMΦ). Design Charles River rats underwent a daily intraperitoneal injection of peritoneal dialysis solution [(PDS), 4.25 g/dL dextrose; Dialine, Travenol, Ashdod, Israel] for a 2-month period (group E). Another group of rats was subjected to the same protocol with the addition of 25 mg/kg aminoguanidine (group A). Three control groups were utilized: ( 1 ) rats that were injected daily with aminoguanidine only (group AO), ( 2 ) rats that were injected with Dulbecco's phosphate-buffered saline (group D), and ( 3 ) rats in which no intervention was carried out (group C). After 2 months, PMΦ were isolated from rat peritoneal effluent and their TNFα production measured by ELISA in cell-free culture supernatants, in both the basal state and after 24-hour stimulation with lipopolysaccharide (LPS). The concentrations of AGEs in peritoneal effluent were assayed and correlated to TNFα levels. PMΦ obtained from normal rats were then incubated for 24 hours with ( 1 ) the peritoneal effluent of each of the above respective groups, with or without LPS; ( 2 ) increasing concentrations of AGEs (0 - 250 μg/mL); and ( 3 ) increasing concentrations of aminoguanidine (0 - 7.5 mg/mL), and TNFα secretion again determined. Results After 2 months of daily intraperitoneal injection of PDS, in the basal state, TNFα production was significantly higher in PMΦ isolated from the peritoneal effluent groups (groups E, A, and AO) compared to controls (group C). Following LPS stimulation, a further increase in TNFα secretion was seen, with a significantly greater response in group AO versus groups E, A, and D. Effluent AGEs were markedly elevated only in group E. No correlation was found between TNFα secretion by these PMΦ and the concentration of AGEs. On incubation with the respective peritoneal effluents (groups E, A, and AO), in both the basal and stimulated state, TNFα production by PMΦ from normal rats was significantly enhanced compared to group C. Incubation with increasing concentrations of AGEs or aminoguanidine resulted in an increase of TNFα secretion by these PMΦ. Conclusions Following intermittent intraperitoneal administration of glucose-based PDS, rat PMΦ are chronically activated, as evidenced by increased basal TNFα secretion. The peritoneal effluent of such treated animals is capable of stimulating TNFα production by normal rat PMΦ. These data suggest that glucose-based PDS acts as a primer of PMΦ, which retain their ability to further stimulation by LPS. Although, in vitro, AGEs promote TNFα secretion by normal rat PMΦ, in vivo, their influence is probably modulated by other factors. Aminoguanidine has a specific inducing effect on rat PMΦ, independent of glucose-based PDS.

Objective Glucose-based dialysate induces non enzymatic glycation within the peritoneal cavity. To evaluate the relationship between the formation of advanced glycation end-products (AGEs) and peritoneal transfer for small solutes and macromolecules, we developed a model of simulated peritoneal dialysis (PD) in normal rats. Methods Male albino rats of the Charles River strain were divided into two sets of 3 groups (15 – 25 rats in each group). In the experimental (E) group, the rats were intra-peritoneally (IP) injected daily with a commercially available 4.25% dextrose solution. In the control puncture (CP) group, the peritoneum was punctured daily, but no PD solution infused. In an age-matched control (CC) group, no intervention was given. Two study protocols were used. Protocol A (duration 20 weeks) consisted of a daily IP injection of 10 mL PD solution per 100 g body weight. In protocol B, a double volume of PD solution was introduced (20 mL per 100 g body weight). At 9, 16, and 20 weeks in protocol A, and at 9 weeks in protocol B, urea, creatinine, microalbumin [(MAL) measured using specific anti-rat albumin monoclonal antibody], and AGEs (measured by fluorescent assay with excitation at 370 nm and emission at 440 nm) were measured in peritoneal effluent and serum. Results At no time during the study were AGEs detected in serum from any group in either protocol. In both protocols, no differences were found between the control groups (CP, CC) with respect to all parameters. In protocol A, the dialysate-to-plasma ratio (D/P) of urea was significantly higher in the experimental group as compared with the control groups at 9, 16, and 20 weeks [9 weeks: 0.59 ± 0.03 (E) vs 0.39 ± 0.02 (CP) vs 0.46 ± 0.02 (CC), p < 0.0004 and p < 0.002, respectively; 16 weeks: 0.71 ± 0.08 (E) vs 0.42 ± 0.01 (CP) vs 0.46 ± 0.01 (CC), p < 0.0001 and p < 0.02, respectively; 20 weeks: 0.57 ± 0.03 (E) vs 0.39 ± 0.01 (CP) vs 0.41 ± 0.02 (CC), p < 0.002 and p < 0.004, respectively]. At 16 and 20 weeks, dialysate MAL levels were significantly increased in group E [16 weeks: 354.00 ± 80.35 μg/mL (E) vs 134.75 ± 14.36 μg/mL (CP) vs 110.69 ± 7.83 μg/mL (CC), p < 0.04 and p < 0.03, respectively; 20 weeks: 283.17 ± 14.71 μg/mL (E) vs 105.14 ± 12.11 μg/mL (CP) vs 135.50 ± 19.03 μg/mL (CC), p < 0.00001 and p < 0.0001, respectively]. In protocol B, at completion of the study (week 9), D/P urea, effluent MAL, and AGEs were significantly higher in the experimental group as compared with the control groups [D/P: 0.67 ± 0.04 (E) vs 0.46 ± 0.07 (CP) vs 0.41 ± 0.02 (CC), p < 0.0002 and p < 00001, respectively; MAL: 336.8 ± 63.30 μg/mL (E) vs 125.71 ± 16.77 μg/mL (CP) vs 119.00 ± 39.75 μg/mL (CC), p < 0.008 and p < 0.007, respectively; AGEs: 265.77 ± 33.49 U/mg creatinine (E) vs 163.10 ± 21.99 U/mg creatinine (CP) vs 83.17 ± 22.66 U/mg creatinine (CC), p < 0.02 and p < 0.001, respectively]. Peritoneal effluent AGEs were found to be significantly correlated with D/P urea and dialysate MAL ( r = 0.42, p < 0.04, and r = 0.7, p = 0.00001, respectively). Conclusions In situ generation of AGEs constitutes the chief origin of peritoneal AGEs. Advanced glycation end-products affect peritoneal permselectivity for both small and large solutes. The rat model of simulated peritoneal dialysis developed in this experiment provides a reliable method for studying peritoneal AGE formation and effect on peritoneal transfer of small solutes and macro-molecules.

Parathyroid hormone (PTH), which is elevated in patients with chronic renal failure, has been shown to participate in the development of vascular calcification. Previous studies have demonstrated that PTH may promote endothelial expressions of proinflammatory parameters. On the basis of these data, we evaluated whether PTH may have an impact on endothelial osteoprotegerin (OPG), a vascular-protective factor which may control vascular calcification. Endothelial cells were stimulated with 10−12to 10−10mol/l PTH. PKC and PKA are the main cellular pathways of PTH. Inhibitors and activators of PKC or PKA were used to determine whether these signaling pathways are involved in the control of endothelial OPG. PTH induced a decrease in OPG secretion and mRNA expression. Treatment of PTH-stimulated cells by calphostin C (PKC inhibitor) induced a further decrease in OPG secretion, while Rp-cAMP (PKA inhibitor) had no additional effect. In nonstimulated cells, a PKC activator significantly stimulated OPG secretion, while a PKA activator was associated with a decline. These effects were blunted in the presence of calphostin C and Rp-cAMP, respectively. An increase in OPG secretion induced by a PKC activator indicates that the basal OPG secretion is mediated through PKC. The decrease induced by a PKA activator, which is similar to the decrease observed with PTH, suggests that the action of PTH on OPG secretion and mRNA expression may be due to the PKA pathway.

Background Disturbances in vitamin D(3) metabolism are associated with an increased cardiovascular morbidity and mortality. The aim of this study was to assess the effects of calcitriol, the active metabolite of vitamin D3, on pro-atherosclerotic parameters in human umbilical vein cord endothelial cells (HUVEC). Materials and methods Calcitriol at 10(-10) and/or 10(-9) mol L(-1) was given to cultured HUVEC which were either non-stimulated or lipopolysaccharide (LPS) stimulated. Inter cellular adhesion molecule-1 and platelet-endothelial cell adhesion molecule-1, were determined by flow cytometry analysis. The receptor of advanced glycation end product (RAGE) and interleukin-6 (IL-6) mRNA expressions by RT-PCR and IL-6 secretion by enzyme-linked immunosorbent assay (ELISA). Nuclear p65 DNA-binding activity was measured by transcription factor assay kit and the inhibitor-kappaBalpha (IkappaBalpha), phosphorylated-IkappaBalpha (P-IkappaBalpha) and phosphorylated-p38 mitogen-activated protein kinase (MAPK) protein levels were determined by Western blot. Results Calcitriol decreased the adhesion molecules expression, as well as the LPS-induced mRNA expressions of RAGE and IL-6 and LPS induced IL-6 secretion. Furthermore, the LPS induced nuclear factor kappaB (NFkappaB)-p65 DNA-binding activity was also decreased by calcitriol. IkappaBalpha levels were increased and p-IkappaBalpha levels decreased after calcitriol treatment. The increased levels of activated p38 MAPK after LPS treatment were also decreased due to pre-incubation with calcitriol. Conclusions The decreased NFkappaB and p38 activities followed by calcitriol treatment may explain the anti-inflammatory/atherosclerotic properties of calcitriol that were observed previously and were emphasized in this study, demonstrating the inhibitory effect of calcitriol on the pro-inflammatory parameters: adhesion molecules, RAGE and IL-6.

Advanced glycation end products (AGEs), which are elevated in diabetic and uremic patients, may induce vascular dysfunctions, and calcitriol may improve the cardiovascular complications. Therefore, we examined whether calcitriol may modify the endothelial response to AGEs stimulation. Knowing the importance of nuclear factor-kappaB in endothelial inflammatory responses, the effect of AGEs and calcitriol on this pathway was also studied. Calcitriol was added to endothelial cells previously incubated with AGE-human serum albumin (HSA). AGE-HSA induced a decrease in endothelial nitric oxide synthase (eNOS) mRNA expression and enzyme activity. Addition of calcitriol to AGE-HSA-treated endothelial cells improved the decreased action of AGEs on the eNOS system. AGE-HSA increased the AGEs receptor mRNA and protein, which were both blunted by calcitriol. The parallel elevation of interleukin-6 mRNA in the presence of AGE-HSA was also blunted by calcitriol. The NF-kappaB-p65 DNA binding activity was enhanced and associated with a decrease in inhibitor kappaBalpha (IkappaBalpha) and an increase in phosphorylated (p)-IkappaBalpha levels. Addition of calcitriol blunted the AGEs-induced elevation of NF-kappaB-p65 DNA binding activity, a phenomenon related to an increased expression of IkappaBalpha. This increase was correlated to declined p-IkappaBalpha levels. The present results support the concept that calcitriol may act as a vascular protective agent counteracting the probable deleterious actions of AGEs on endothelial cell activities.

Complete remission can be achieved in 60-80% of adults with diffuse aggressive non-Hodgkin's lymphoma. However, 20-40% of them will subsequently relapse. Nevertheless, formal follow-up guidelines for recurrence detection have never been advocated. We analyzed the pattern of relapse in 30 patients with intermediate- and high-grade non-Hodgkin's lymphoma and the value of intensive protocol for relapse detection. This protocol includes frequent follow-up visits, complete blood count, and serum LDH tests along with annual chest, abdominal, and pelvic CT scans. The median duration of complete remission was 12 months. Twenty-five relapses (83%) were suspected after an interim history and/or physical examination, whereas only 5 relapses (17%) were detected by routine radiographic or laboratory follow-up studies. The majority of relapses (19/30) were detected in sites that included the sites of prior disease. For the first 12 months of complete remission, the estimated cumulative save in charge for a follow-up strategy, based on regular visits in the hematology clinic and performing laboratory and radiologic studies as clinically indicated, is 44% of the cost of a routine intensive evaluation. A reliable and cost-effective follow-up method for non-Hodgkin's lymphoma patients in complete remission should include frequent history and physical examination. Complementary studies should be performed according to clinical indications.