Shifra Sela

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.

Participation of Peripheral Polymorphonuclear Leukocytes in the Oxidative Stress and Inflammation in Patients with Essential Hypertension

Oxidative stress and inflammation have recently been linked to endothelial damage in essential hypertension (EH). Activated peripheral polymorphonuclear leukocytes (PMN) damage surrounding tissue by releasing reactive oxygen species (ROS) and proteolytic enzymes before self-necrosis. PMN necrosis further exacerbates inflammation and promotes chemotaxis and PMN recruitment. The number and properties of PMN from untreated EH patients is the focus of the present study. Oxidative stress was assessed by measuring the rate of superoxide anion release from separated, phorbol ester-stimulated PMN and the redox state of plasma glutathione. Inflammation was estimated indirectly by determining PMN number and their in vitro survival. PMN from EH patients (n = 37) released superoxide anion faster (P < .0001) than those of normotensives (NC, n = 37), 17.7 +/- 1.14 v 9.54 +/- 0.51 nmol/10 min/10(6) cells. The redox state of glutathione was twofold higher in EH plasma (P < .02) indicating systemic oxidative stress. PMN survival in vitro correlates linearly with the rate of superoxide release (r2 = 0.60, P < .02) and PMN count of EH patients, although in the normal range, were significantly higher (P < .0001), indicating necrosis and recruitment. Hypertensive plasma significantly reduced NC PMN viability, whereas normal plasma significantly increased EH PMN viability. What our studies show is that EH is accompanied by a primed state PMN that does not correlate with the levels of blood pressure. PMN priming in EH patients reflects an in vivo exposure to a constant stimulus ending in oxidative stress, increased self-necrosis, and cell recruitment. Oxidative stress and inflammation will result in endothelial damage and atherosclerosis in the long run.

The polymorphonuclear leukocyte--a new target for erythropoietin.

A previous study from our laboratory has shown that erythropoietin (EPO), beside its traditional role in erythropoiesis, acts as an alleviator of oxidative stress and inflammation in chronic hemodialysis (HD) patients, conferred in part by activated polymorphonuclear leukocytes (PMNLs). To substantiate this phenomenon, the existence of EPO receptors (EPO-Rs) on PMNL membrane was examined at the transcriptional and translational levels. mRNA for EPO-R was detected in PMNLs using specific primers directed towards the extracellular region of human EPO-R cDNA. The predicted 300-bp fragment was amplified by reverse transcriptase-polymerase chain reaction. Subcloning and sequence analysis revealed 100% homology of this fragment with human EPO-R. The receptor protein was detected on the surface of intact PMNLs using 125I-EPO. The protein was further demonstrated by flow cytometric analysis using a fluorescent monoclonal anti-EPO-R. The percentage of PMNLs expressing EPO-R showed a strong correlation with the level of EPO in the serum, suggesting an upregulation of the receptor by the hormone. Taken together with our recent findings that EPO attenuates the oxidative stress and inflammation contributed by PMNLs in HD patients, the detection of functional EPO-R expression in PMNLs places these cells among the nonerythroid, EPO-responsive target populations.

Renal Ischemia – Reperfusion Injury: Contribution of Nitric Oxide and Renal Blood Flow

The contributions of nitric oxide (NO) and renal blood flow (RBF) were examined in ischemia-reperfusion injury in the rat kidney. The function of both kidneys was assessed by glomerular filtration rate (GFR), and fractional excretion of sodium (FENa), calculated before, during unilateral renal artery clamping (45 min), and following reperfusion (90 min). RBF was measured in the same model by ultrasonic flowmetry. Intrarenal NO levels were modulated by administration of S-nitroso-N-acetylpenicillamine (SNAP), L-arginine, acetylcholine, and the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). SNAP increased GFR from 0.20 ± 0.04 ml/min in control ischemic kidney to 0.38 ± 0.06 ml/min and reduced FENa from 19.3 ± 3.4 to 9.5 ± 1.8%. Similar results were observed when L-arginine was administered. Acetylcholine had no effect on GFR or FENa. RBF was fully restored within 60 min following reperfusion, with no change in the rate of recovery by L-arginine. L-NAME aggravated the ischemia-reperfusion injury, preventing full restoration of RBF, further reducing GFR and worsening FENa. In conclusion, ischemia-reperfusion injury ends in low intrarenal levels of NO. We propose that this low NO level results from damage to the endothelial receptor signal transduction process and is not due to impaired NO synthase activity or to changes in RBF.

Interaction between Erythropoietin and Peripheral Polymorphonuclear Leukocytes in Hemodialysis Patients

The effect of erythropoietin (EPO) on the oxidative stress and inflammation caused by polymorphonuclear leukocytes (PMNLs) in chronic hemodialysis (HD) patients was investigated in vivo and in vitro. The studies were performed on isolated PMNLs from peripheral blood of healthy controls and HD patients before and following 6 weeks of EPO treatment. The oxidative stress was expressed by the rate of superoxide release from phorbol 12-myristate 13-acetate stimulated PMNLs, and the inflammatory state was evaluated by in vitro PMNL survival, in addition to white blood cell and PMNL counts of the enrolled subjects. Following 6 weeks of EPO treatment, in HD patients, the rate of superoxide release from PMNLs as well as WBC and PMNL counts fell significantly when compared with the pretreatment values. PMNLs from HD patients and healthy controls incubated in vitro with increasing amounts of EPO displayed a significant reduction in their rates of superoxide release and a significant improvement in survival. We have concluded that EPO interacts with PMNLs, attenuating their primed state in HD patients, thus reducing oxidative stress and the extent of inflammation. To the best of our knowledge, this attenuation of the primed state of PMNLs by EPO is a new finding.

Primed Polymorphonuclear Leukocytes, Oxidative Stress, and Inflammation Antecede Hypertension in the Sabra Rat

Hypertension is accompanied by systemic oxidative stress, inflammation, and priming of peripheral polymorphonuclear leukocytes (PMNLs), yet the involvement of these factors in the pathophysiology of hypertension is incompletely understood. We investigated the relationship between oxidative stress, primed PMNLs, and inflammation and the development of hypertension in the Sabra rat model of salt-sensitive hypertension. Sabra hypertension-resistant rats (SBN/y) (salt-resistant) and Sabra hypertension-prone rats (SBH/y) (salt-sensitive) were studied under normal conditions or during salt loading. Systolic blood pressure (BP) was measured by the tail-cuff method. The extent of oxidative stress was evaluated by the rate of superoxide release from PMNLs, plasma-reduced glutathione (GSH) levels, malondialdehyde (MDA) levels (estimated by thiobarbituric acid–reacting substances), and plasma-carbonylated fibrinogen (Western blotting). Plasma fibrinogen levels and the peripheral PMNL count served as indices of inflammation. In SBH/y and SBN/y provided regular chow without salt loading, BP did not rise above baseline values, yet superoxide release, plasma MDA, carbonylated fibrinogen, and PMNL count were higher in SBH/y than in SBN/y, whereas GSH levels were lower in SBH/y. Four weeks of salt loading resulted in a gradual increase in systolic BP in SBH/y to 205±3 mm Hg, whereas BP remained in SBN/y at baseline normotensive levels. All the parameters reflecting oxidative stress and inflammation were further aggravated with the development of hypertension in salt-loaded SBH/y. We conclude that primed PMNLs, oxidative stress, and inflammation antecede the development of hypertension in this experimental model of hypertension.

Effect of oxygen tension on activity of antioxidant enzymes and on renal function of the postischemic reperfused rat kidney.

The aim of the present study was to examine the effect of exposing animals to 100% oxygen instead of room air on renal function and endogenous antioxidant enzymes of the postischemic reperfused rat kidney. Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were determined in the homogenate of the left kidney after 45 min of ischemia, caused by clamping the left renal artery, 10 and 90 min after reperfusion while the animals breathed room air or 100% O2. The right kidney served as a control. The possible influence of trapped blood in the clamped kidney was also investigated by the use of a correction factor based on the Hb concentration in the homogenate. The results indicate that such correction is necessary as the blood adds significant antioxidant activity. The activities of all 3 enzymes after 45 min of ischemia decreased significantly in the left (ischemic) compared to the right (control) kidney, to 64% of the control levels for catalase, 58% for SOD and 49% for GPX. After 10 min of reflow, a further decrease in the activities of catalase (to 49%) and of GPX (to 29%) was found. SOD activity, however, increased to 64%. After 90 min of reperfusion, restoration toward normal levels was noticed (SOD activity increased to 70%, catalase to 76% and GPX to 58%). Breathing 100% O2 resulted in a significant decrease in all enzyme activities (to 38.6% for catalase, 45% for SOD and to 27.4% for GPX). This inactivation can be explained by increased reactive oxygen species (ROS) activity during hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Salivary β2-Microglobulin Analysis in Chronic Kidney Disease and Hemodialyzed Patients

Background: β2-Microglobulin (β2m) is a major component in dialysis-related amyloidosis, a disabling disease affecting long-term dialysis patients. Methods: β2m and other components were analyzed in saliva and serum from 53 individuals in 4 subgroups: healthy normal controls, diabetes mellitus (DM), chronic kidney disease (CKD) and hemodialysis (HD) patients. Results: Median salivary and mean serum β2m concentrations were 78% higher in both saliva (p = 0.048) and serum (p = 0.047) in DM patients; 118% (p = 0.016) and 907% (p = 0.007) higher, respectively, in CKD patients, and 328% (p = 0.0001) and 2,710% (p = 0.001) higher, respectively, in HD patients, compared with healthy controls. The correlation analysis between salivary and serum β2m concentrations showed a low correlation rate in HD patients (r = –0.18), but was rather high in CKD patients (r = 0.50). Conclusions: Salivary analysis of β2m is a reliable method for evaluating serum β2m levels in CKD patients, and may help predict the potential for development of CKD-induced amyloidosis.

Heparin cryoprecipitation reduces plasma levels of non-traditional risk factors for atherosclerosis in vitro.

Aims: To show that heparin cryoprecipitation (HCP), an in vitro method of plasma purification, reduces the levels of in vivo modified proteins and non-traditional risk factors from plasma of atherosclerotic hemodialysis (HD) patients. Methods: HCP was applied to plasma obtained from HD patients and controls, forming a precipitate – cryogel. Levels of fibrinogen, albumin, CRP, TNF-α, IL-6, advanced oxidation protein products, carbonylated fibrinogen and carbonylated albumin were determined in plasma before and after applying HCP and in the cryogel. Results: Treatment of HD plasma with HCP, beyond the significant reduction of the increased levels of all the above-mentioned molecules, reduced fibrinogen, TNF-α, carbonylated fibrinogen and carbonylated albumin to control levels which were simultaneously found in the cryogel. Conclusions: HCP applied to plasma enables the simultaneous precipitation of modified molecules and circulating non-traditional risk factors for atherosclerosis. This study may serve as a base for the future development of a clinical purification technique.