[LEVEL AND OXIDATION OF BETA 2-MICROGLOBULIN IN HEMODIALYSIS PATIENTS TREATED WITH INTRAVENOUS IRON DURING DIALYSIS WITH HIGH-FLUX COMPARED TO LOW-FLUX DIALYZERS].

INTRODUCTION: Serum levels of ß2-microglobulin (b2M) are significantly higher in patients with end stage renal failure undergoing hemodialysis (HD) and its accumulation accelerates Dialysis Related Amyloidosis (DRA). In HD patients low-flux dialysis, intravenous (IV) iron (administered for the treatment of anemia) affects ß2M removal during dialysis. IV iron also affects the oxidation of plasma proteins, including b2M. AIMS: To examine the effect of intravenous iron therapy on ß2M levels and oxidation in HD patients treated with high-flux compared with low-flux dialyzers. METHODS: Sixteen HD patients on chronic maintenance IV iron therapy were studied. Half of the patients were allocated to high-flux and half to low-flux dialysis. After five weeks, each patient was assigned to the second dialyzer. After two weeks of treatment with each dialyzer, blood samples were taken and serum levels of ß2M were measured. In addition, the hematocrit and iron status were measured. Part of the samples were used to evaluate oxidized ß2M. RESULTS: A significant increase in ß2M levels was found with low-flux dialysis, which further increased during dialysis with IV iron administration. High-flux dialysis therapy significantly lowered the ß2M levels, with a clear decrease during the dialysis session, that was unaffected by IV iron administration. A significant decrease in ß2M oxidation was found during highflux, but not low-flux dialysis. CONCLUSIONS: High-flux dialysis is more effective than lowflux in decreasing the levels and oxidation of ß2M. These observations may have significance in improving iron therapy, aimed to decrease or attenuate the appearance of DRA.

Lercanidipine effect on polymorphonuclear leukocyte-related inflammation and insulin resistance in essential hypertension patients.

INTRODUCTION: Inflammation, insulin resistance, and oxidative stress (OS) are among the mechanisms that have been implicated in the pathogenesis of essential hypertension (EH). Peripheral polymorphonuclear leukocytes (PMNLs) are primed in EH patients, releasing uncontrolled superoxide anions contributing to OS in these patients. PMNL priming correlates with insulin resistance and PMNL intracellular calcium ([Ca(2+)]i). Recent studies have attributed additional anti-ischemic and antioxidative characteristics to the antihypertensive drug, lercanidipine, a third-generation calcium-channel blocker. The purpose of this study was to evaluate the possible nontraditional effect of 2 months of lercanidipine treatment on insulin resistance and on PMNL-related inflammation in EH patients. METHODS: Non-smoking EH patients with untreated mild-to-moderate high blood pressure (BP) were included. Low-grade inflammation was reflected by PMNL apoptosis and by white blood cell (WBC) and PMNL counts. Systemic inflammation was measured by plasma fibrinogen, C-reactive protein (CRP), and transferrin and albumin levels. Fasting serum insulin levels served as a marker of insulin resistance. RESULTS: Two months of lercanidipine treatment showed a significant decrease in BP, WBC, and PMNL counts, PMNL apoptosis, CRP, and serum insulin levels, and a significant increase in serum albumin levels. Rates of superoxide release from PMNLs, WBC and PMNL counts, and insulin levels positively correlated with mean arterial BP values. CONCLUSION: The use of lercanidipine can be favorable in EH patients due to its combined anti-PMNL priming and anti-inflammatory effects, in addition to its antihypertensive characteristics.

High blood pressure response to stress ergometry could predict future hypertension.

BACKGROUND: Previous studies have shown that exaggerated blood pressure (BP) during exercise is a valid risk predictor for future hypertension in most men and women, yet the use of ergometry as a means of early detection of incipient hypertension still requires confirmation. OBJECTIVES: To assess the clinical utility of exercise BP measurement for the evaluation of risk for developing new-onset hypertension. METHODS: Thirty individuals with normal BP were enrolled in this study and were subsequently divided into two groups: 13 persons with in-exercise hypertension were compared with 17 matched persons who were normotensive during ergometry. Their blood pressure was monitored during follow-up of two years. RESULTS: More individuals in the exercise-hypertensive group developed hypertension after one or two years than those normotensive during the exercise (respectively, one year: 3 vs.0, p=0.03, two years: 10 vs. 1, p<0.0001). Both the systolic and diastolic BPs significantly differed between the two groups. Eighty four percent of those with exaggerated BP (>or=210 mm Hg) during the treadmill exercise developed hypertension after 2 years. The sensitivity and specificity of in-exercise hypertension for predicting its 2 year occurrence were, respectively, 91% and 84%. CONCLUSION: Even in the absence of hypertension, its development during stress ergometry could be considered a predictive marker for the future development of hypertension, and can be a potential tool for identifying normotensive individuals at high risk. These individuals should be followed up and their BP controlled for a long time.

Intensification of oxidative stress and inflammation in type 2 diabetes despite antihyperglycemic treatment.

INTRODUCTION: The metabolic deregulation associated with diabetes mellitus (DM) causes secondary pathophysiologic changes in multiple organ systems. Endothelial injury is induced by oxidative stress (OS) and inflammation. We have previously shown that DM type 2 patients are exposed to increased OS and inflammation contributed in part by primed peripheral polymorphonuclear leukocytes (PMNLs). AIMS: To characterize the effect of oral medication on PMNL priming, on PMNL-related and on systemic inflammation, in correlation to changed diabetes parameters in patient with newly diagnosed type 2 DM. METHODS: PMNLs were separated from DM patient's prior and following treatment with either metformin (Glucophage), or Thiazolidinedione (rosiglitazone) and from healthy control subjects (HC). Rate of superoxide release from phorbol ester-stimulated PMNLs and CD11b on PMNLs assessed PMNL priming. White blood cells (WBC) and PMNL counts and apoptosis reflected PMNL-related inflammation. CRP, fibrinogen, transferrin and albumin blood levels reflected systemic inflammation. RESULTS: Both metformin and rosiglitazone treatments reduced significantly the high levels of glucose and HbA1c, and slightly improved lipid profile during 2 months. PMNL priming parameters, higher compared to HC, increased after 2 months of metformin treatment. Rosiglitazone treatment decreased PMNL priming. ALP, higher in DM, significantly decreased following 2 months of both treatments. Systemic inflammation markers (fibrinogen, CRP), higher in DM, decreased following both treatments. Transferrin and albumin were similar to HC. PMNL-related inflammation markers were higher in DM; however, only PMNL apoptosis decreased after both treatments. Monocyte counts, higher in DM compared to HC, decreased following both treatments. Serum insulin levels, higher in DM compared to HC, decreased following both treatments. PMNL-related priming and inflammation parameters positively correlated with HbA1c. CONCLUSION: The present research adds new facet in evaluating anti-hyperglycemic treatment in type 2 DM patients. Despite sufficient glycemic control using both treatments, some PMNL-related parameters deteriorated. Thus, anti hyperglycemic treatment should be favored due to its combined anti-PMNL priming and anti-inflammatory effect, in addition to its anti-hyperglycemic characteristics, according to the correlation among these parameters. Such combined treatment may reduce morbidity and mortality common in DM patients.

Epoetin-alpha: preserving kidney function via attenuation of polymorphonuclear leukocyte priming.

BACKGROUND: Polymorphonuclear leukocyte priming and low grade inflammation are related to severity of kidney disease. Erythropoietin-receptor is present on PMNLs. OBJECTIVESxi: To evaluate the effect of 20 weeks of epoetin-alpha treatment on PMNL characteristics in relation to the rate of kidney function deterioration in patients with chronic kidney disease. METHODS: Forty anemic chronic kidney disease patients, stage 4-5, were assigned to EPO and non-EPO treatment for 20 weeks. A group of 20 healthy controls was also studied. PMNL priming and PMNL-derived low grade inflammation were estimated, in vivo and ex vivo, before and after EPO treatment: The rate of superoxide release, white blood cells and PMNL counts, serum alkaline phosphatase and PMNL viability were measured. EPO-receptor on PMNLs was assayed by flow cytometry. The effect of 20 weeks of EPO treatment on kidney function was related to the estimated glomerular filtration rate. esults: EPO treatment attenuated superoxide release ex vivo and in vivo and promoted PMNL survival ex vivo. Decreased low grade inflammation was reflected by reduced WBC and PMNL counts and ALP activity following treatment. EPO retarded the deterioration in GFR. The percent of PMNLs expressing EPO-R was higher before EPO treatment and correlated positively with the rate of superoxide release. After 20 weeks of EPO treatment the percent of PMNLs expressing EPO-R was down-regulated. CONCLUSIONS: These non-erythropoietic properties of EPO are mediated by EPO-R on PMNLs, not related to the anemia correction. A new renal protection effect of EPO via attenuation of PMNL priming that decreases systemic low grade inflammation and oxidative stress is suggested.

Primed polymorphonuclear leukocytes constitute a possible link between inflammation and oxidative stress in hyperlipidemic patients.

BACKGROUND: Oxidative stress (OS) and chronic inflammation are involved and contribute to the development of atherosclerosis. Primed polymorphonuclear leukocytes (PMNLs) are a possible source for superoxide radicals and inflammatory mediators, hence can promote OS and inflammation. The involvement of primed PMNLs in clinical states associated with high risk for developing cardiovascular disease and atherosclerosis, such as hypertension, renal failure and diabetes has been described, however, little is known about PMNLs characteristics in hyperlipidemic patients. METHODS: Hyperlipidemic patients and healthy control (HC) subjects were enrolled in this cross-sectional study. PMNL priming was estimated by measuring the rate of superoxide release and by levels of membrane CD11b. PMNL priming and myeloperoxidase (MPO) levels served as OS indices. Inflammation was linked to peripheral white blood cells and PMNL counts and to apoptosis. Systemic inflammation was estimated by blood levels of fibrinogen, C-reactive protein (CRP), transferrin and albumin. PMNL priming and inflammation parameters were related to the severity of hyperlipidemia. RESULTS: PMNLs from hyperlipidemic patients are primed compared to HC. A decrease in PMNL-MPO levels with increased levels of serum MPO were found in hyperlipidemic patients. Leukocyte counts tended to be higher in hyperlipidemic patients with increased PMNL apoptosis. PMNL priming and fibrinogen levels correlated positively with the severity of hyperlipidemia (r=0.32, P=0.02 for CD11b vs. cholesterol and r=0.38, P=0.009 for CD11b vs. LDL-c; r=0.35, P=0.01 for fibrinogen vs. cholesterol and r=0.3, P=0.03 for superoxide release vs. LDL-c). CONCLUSION: PMNLs are primed in hyperlipidemic patients contributing to OS and inflammation in these patients. This study highlights primed PMNLs as an additional risk factor for promoting atherosclerosis in hyperlipidemic patients.

Polymorphonuclear leucocyte priming in long intermittent nocturnal haemodialysis patients--is melatonin a player?

BACKGROUND: The relationships between sleep quality, melatonin circadian rhythm and polymorphonuclear leucocyte (PMNL) priming during the night of dialysis treatment compared with a night without dialysis were studied in a group of nocturnal haemodialysis (HD) patients. METHODS: Twenty-eight long intermittent nocturnal HD patients were included. Sleep quality was assessed by a questionnaire and wrist actigraphy. Plasma melatonin levels were assayed every 2 h, from 9 p.m. to 5 a.m. PMNL priming was assessed by the rate of superoxide release from separated PMNLs at 9 p.m. and 5 a.m., on a night of dialysis and a night with sleepover in the dialysis unit without being dialysed. RESULTS: Melatonin levels increased similarly during a night with and without dialysis, reaching peak level at 5 a.m. Most (73%) of the patients had severe sleep disturbances. A significant negative correlation was found between the sleep quality score, the rate of superoxide release from separated PMNLs and melatonin levels. While during a night without dialysis a significant reduction of the rate of superoxide release was found at 5 a.m. (compared with 9 p.m.), no significant reduction was observed when the patients were dialysed. Patients with flat melatonin curves, with <10 pg/ml, showed a faster rate of superoxide release than those with higher levels. CONCLUSIONS: The nocturnal HD process does not affect plasma melatonin levels or rhythms, suggesting that melatonin is not dialysed. Higher endogenous melatonin levels are associated with better sleep and lower PMNL priming. The lower PMNL priming in patients with higher plasma melatonin levels suggests that melatonin overrides the oxidative burden induced by the dialysis process.

[The significance of troponin T in hemodialysis patients].

INTRODUCTION: Troponin T and I are located in the myocardium. Monitoring blood Troponin levels is advantageous in evaluating unstable angina over measuring CPK-MB, previously serving as a "golden standard" for urgent evaluation of myocardial infarct. AIM: To measure blood troponin T levels in hemodialysis patients without severe cardiovascular events, following its distribution according to various risk factors. METHODS: Sera were collected from 68 hemodialysis (HD) patients before starting dialysis sessions and compared to 10 age- and gender-matched healthy volunteers (NC). Troponin T levels were measured using Troponin T STAT Elecsys-Roche kits. RESULTS: Blood Troponin T levels were significantly higher in HD patients compared to NC (0.126 +/- 0.016 vs. 0.005 +/- 0 ng/dL, respectively; P=0.026). HD patients with ischemic heart disease (IHD) showed significantly higher levels of Troponin T, compared to HD patients without IHD (0.17+/-0.036 vs. 0.081 +/- 0.015 ng/dL, respectively; P=0.012). Diabetic (DM) HD patients showed higher levels of Troponin T, compared to non-DM HD patients (0.168+/-0.034 vs. 0.068+/-0.012 ng/dL, respectively; P= 0.002). CONCLUSION: Troponin T may efficiently serve as a diagnostic, monitoring and prognostic parameter in HD patients.

[The effect of calcium channel blocker lercanidipine on lowgrade inflammation parameters in essential hypertension patients].

INTRODUCTION: Oxidative stress (OS), inflammation and insulin resistance are among the mechanisms that have been recently implicated in pathogenesis of essential hypertension (EH). Peripheral polymorphonuclear leukocytes (PMNLs) are primed in EH patients, releasing uncontrolled superoxide anion contributing to OS and chronic low-grade inflammation in these patients. PMNL priming correlates with insulin resistance and with PMNL intracellular calcium ([Ca2+]i). Recent studies have attributed additional anti-oxidative characteristics to the anti-hypertensive drug Lercanidipine (Vasodip), a third generation calcium-channel blocker. AIM: To evaluate possible novel effects of two months of Lercanidipine treatment on systemic and PMNL-related inflammation and on insulin resistance in EH patients. METHODS: Fifteen non-smoking EH patients with untreated mild to moderate high blood pressure (BP) and age- and gender-matched healthy controls (HC) were included in the study. Low-grade inflammation was expressed by PMNL counts and apoptosis, by plasma fibrinogen, CRP and albumin (as a negative acute phase reactant) levels. Fasting serum insulin levels served as a marker of insulin resistance. RESULTS: Inflammation parameters and insulin levels were higher in EH compared to HC. PMNL counts, fibrinogen and insulin levels positively correlated with mean arterial blood pressure values. Two months of Lercanidipine treatment showed a significant decrease in BP, PMNL counts and apoptosis, CRP and serum insulin levels and a significant increase in serum albumin levels. CONCLUSION: The authors imply that the low-grade systemic inflammation and insulin resistance detected in EH patients may be attenuated by the use of Lercanidipine, adding new unknown anti-inflammatory properties to this calcium channel blocker.

[Polymorphonuclear leukocyte priming and counts are in correlation with blood pressure parameters].

BACKGROUND: In hypertensive patients, the polymorphonuclear leukocytes (PMNLs) are primed, concomitantly contributing to oxidative stress and chronic low-grade inflammation. Furthermore, in the Sabra rat model of salt-induced hypertension, priming of PMNLs, oxidative stress and inflammation antecede the development of hypertension. In the present study we tested the hypothesis that PMNL priming and PMNL and white blood cells (WBC) counts are interrelated with blood pressure values. Therefore, we have evaluated the correlation between WBC and PMNL counts, PMNL priming parameters and blood pressure in untreated essential hypertension patients (EH) and age and gender healthy controls. METHODS: Diastolic blood pressure (DBP), systolic blood pressure (SBP) and mean arterial pressure (MAP) values were correlated by linear regression analysis with the rates of superoxide release from separated PMNLs and with WBC and PMNL counts. RESULTS: The rate of superoxide release from PMNLs was higher in EH patients compared to their healthy controls. The rate of superoxide release from PMNLs correlated with SBP, DBP and MAP. WBC and PMNL counts were all significantly correlated with blood pressure values. CONCLUSION: The results of the study offer an additional mechanism involving primed PMNLs and elevated PMNL counts in the pathogenesis of hypertension and its late cardiovascular complications.

Primed peripheral polymorphonuclear leukocyte: a culprit underlying chronic low-grade inflammation and systemic oxidative stress in chronic kidney disease.

This study characterizes the causal relationship between peripheral polymorphonuclear leukocyte (PMNL) priming, systemic oxidative stress (OS), and inflammation in patients with varying degrees of renal insufficiency (chronic kidney disease [CKD] not on renal replacement therapy [RRT]: continuous ambulatory peritoneal dialysis or hemodialysis [HD]) and healthy control subjects. Rate of superoxide release was measured after stimulation of PMNL with phorbol 12-myristate 13-acetate or zymosan. Priming was estimated by the rate of superoxide release after phorbol 12-myristate 13-acetate stimulation. Systemic OS was related to PMNL priming and intracellular myeloperoxidase activity. Inflammation was linked to peripheral white blood cells and PMNL counts, PMNL apoptosis, and PMNL ex vivo survival in autologous and heterologous sera. PMNL priming and counts were related to the severity of renal failure in CKD not on RRT. Compared with control subjects, PMNL from all CKD patients showed increased priming, highest in HD, with a significant decrease in their response to zymosan. PMNL myeloperoxidase activity and apoptosis were increased in all renal failure patients. Decreased ex vivo cell survival and elevated leukocyte counts were found in all patients, highest in HD. Both PMNL priming and counts correlated negatively with the GFR. A positive significant correlation was shown between PMNL counts and their priming in all groups, suggesting that the increased PMNL count in peripheral blood is an adaptive response to PMNL priming. Hence, PMNL priming is a key mediator of low-grade inflammation and OS associated with renal failure, occurring before the onset of RRT and further augmented in chronic HD.

Carbonyl stress induced by intravenous iron during haemodialysis.

BACKGROUND: Anaemic haemodialysis (HD) patients are treated with erythropoietin and intravenous iron for effective erythropoiesis. Since iron is a potent inducer and aggravator of pre-existing oxidative processes in HD patients, this study was aimed to evaluate the acute in vivo effect of two recommended iron doses on protein oxidation during the HD session. METHODS: Iron gluconate was intravenously administered to HD patients in doses of 62.5 or 125 mg per session. A dialysis session without iron administration served as a control for each patient. Carbonylated fibrinogen and iron profile parameters were monitored before and after each session. Plasma carbonylated fibrinogen levels from healthy subjects and HD patients before dialysis were compared. Protein associated carbonyls were identified in plasma by derivatization with 2,4-dinitrophenylhydrazine followed by western analysis and were quantified by densitometry. RESULTS: HD patients on maintenance iron showed elevated carbonylated fibrinogen compared with healthy subjects. During a HD session, carbonyls on fibrinogen further increased when 125 mg iron gluconate was administered, but no changes were detected with 62.5 mg iron gluconate or in the absence of iron. The changes in carbonylated fibrinogen during dialysis showed a significant linear correlation with the calculated values of transferrin saturation and free transferrin. CONCLUSIONS: The significant acute increase in carbonylated fibrinogen with 125 mg iron gluconate suggests that this iron dose should be used with caution. As fibrinogen is highly susceptible to iron-induced oxidation in vivo, it may serve as a marker reflecting acute iron oxidative damage and as a tool in refinement of the existing clinical dose guidelines for intravenous iron therapy.

The involvement of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation among cigarette smokers.

BACKGROUND: Cigarette smoking is a well-known risk factor for the development of endothelial dysfunction and the progression of atherosclerosis. Oxidative stress and inflammation have recently been implicated in endothelial dysfunction. OBJECTIVES: To assess the concomitant contribution of polymorphonuclear leukocytes to systemic oxidative stress and inflammation in cigarette smokers. METHODS: The study group comprised 41 chronic cigarette-smoking, otherwise healthy males aged 45.0 +/- 11.5 (range 31-67 years) and 41 male non-smokers aged 42.6 +/- 11.3 (range 31-65) who served as the control group. The potential generation of oxidative stress was assessed by measuring the rate of superoxide release from separated, phorbol 12-myristate 13-acetate-stimulated PMNL and by plasma levels of reduced (GSH) and oxidized (GSSG) glutathione. Inflammation was estimated indirectly by: a) determining the in vitro survival of PMNL, reflecting cell necrosis; b) in vivo peripheral PMNL counts, reflecting cell recruitment; and c) plasma alkaline phosphatase levels, indicating PMNL activation and degranulation. RESULTS: PMA-stimulated PMNL from cigarette smokers released superoxide at a faster rate than PMNL from the controls. Smokers had decreased plasma GSH and elevated GSSG levels. In vitro incubation of control and smokers' PMNL in sera of smokers caused necrosis, while control sera improved smoker PMNL survival. Smokers' PMNL counts, although in the normal range, were significantly higher than those of controls. Plasma ALP levels in smokers were significantly higher than in controls and correlated positively with superoxide release and PMNL counts. CONCLUSIONS: Our study shows that PMNL in smokers are primed in vivo, contribution concomitantly to systemic oxidative stress and inflammation that predispose smokers to endothelial dysfunction, and explains in part the accelerated atherosclerosis found in smokers.

Interaction between erythropoietin and peripheral polymorphonuclear leukocytes in continuous ambulatory dialysis patients.

The effect of erythropoietin (EPO) on the oxidative stress (OS) and inflammation caused by polymorphonuclear leukocytes (PMNLs) in end-stage renal failure patients undergoing continuous ambulatory peritoneal dialysis (CAPD) was investigated in vivo and in vitro. The studies were performed on isolated PMNLs from peripheral blood of CAPD patients before and following 6 weeks of EPO treatment and from healthy controls. OS was expressed by the rate of superoxide release from phorbol 12-myristate 13-acetate (PMA) stimulated isolated PMNLs and the inflammatory state was evaluated by PMNL counts of the enrolled subjects. Following 6 weeks of EPO treatment in CAPD patients, both the rate of superoxide release from PMNLs and PMNL counts fell significantly when compared with the pretreatment values. In vitro incubation of PMNLs from CAPD patients with increasing amounts of EPO displayed a significant reduction in their rates of superoxide release. EPO, by direct interaction with PMNLs, attenuated their primed state, causing reduction in oxidative stress and inflammation.

A link between polymorphonuclear leukocyte intracellular calcium, plasma insulin, and essential hypertension.

BACKGROUND: Intracellular ionized calcium ([Ca2+]i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca2+]i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca2+]i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP). METHODS: The PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca2+]i was measured by flow cytometry in isolated cells stained with Fluo-3. RESULTS: The EH PMNs showed significantly increased [Ca2+]i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca2+]i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca2+]i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04). CONCLUSIONS: This study adds PMN to previously described cells exhibiting elevated [Ca2+]i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca2+]i and plasma insulin levels, together with the fact that elevated [Ca2+]i mediates PMN priming, suggest that elevated [Ca2+]i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.