* Eliachar Research Laboratory; Department of Nephrology; Department of Hematology, Western Galilee Hospital, Nahariya; and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel
Address correspondence to: Dr. Batya Kristal, Head of Nephrology and Hypertension Department, Western Galilee Hospital, Nahariya 22100, Israel. Phone: 972-4-9107603; Fax: 972-4-9107482, 972-4-9107469; E-mail: batya.kristal{at}naharia.health.gov.il
Received for publication November 11, 2004.
Accepted for publication April 28, 2005.
This study characterizes the causal relationship between peripheralpolymorphonuclear leukocyte (PMNL) priming, systemic oxidativestress (OS), and inflammation in patients with varying degreesof renal insufficiency (chronic kidney disease [CKD] not onrenal replacement therapy [RRT]: continuous ambulatory peritonealdialysis or hemodialysis [HD]) and healthy control subjects.Rate of superoxide release was measured after stimulation ofPMNL with phorbol 12-myristate 13-acetate or zymosan. Primingwas estimated by the rate of superoxide release after phorbol12-myristate 13-acetate stimulation. Systemic OS was relatedto PMNL priming and intracellular myeloperoxidase activity.Inflammation was linked to peripheral white blood cells andPMNL counts, PMNL apoptosis, and PMNL ex vivo survival in autologousand heterologous sera. PMNL priming and counts were relatedto the severity of renal failure in CKD not on RRT. Comparedwith control subjects, PMNL from all CKD patients showed increasedpriming, highest in HD, with a significant decrease in theirresponse to zymosan. PMNL myeloperoxidase activity and apoptosiswere increased in all renal failure patients. Decreased ex vivocell survival and elevated leukocyte counts were found in allpatients, highest in HD. Both PMNL priming and counts correlatednegatively with the GFR. A positive significant correlationwas shown between PMNL counts and their priming in all groups,suggesting that the increased PMNL count in peripheral bloodis an adaptive response to PMNL priming. Hence, PMNL primingis a key mediator of low-grade inflammation and OS associatedwith renal failure, occurring before the onset of RRT and furtheraugmented in chronic HD.
The polymorphonuclear leukocyte (PMNL), one of the main inflammatorycell types, exists in the blood stream in one of three functionalstates: Quiescent, primed, or activated (1). Under noninfectiousconditions, the PMNL are quiescent, exhibiting little or norelease of reactive oxygen species (ROS). Studies have led tothe concept of a two-stage activation process: PMNL first encountera stimulus that leaves the cells in a "primed" state. Upon encounteringa second stimulus, PMNL proceed to the second state of fullactivation, releasing ROS, granule contents, and inflammatorymediators (1–3). Ward and McLeish (4–6) reportedthat PMNL from patients with chronic kidney disease (CKD) bothbefore and while on renal replacement therapy (RRT) are primed.Our studies have also shown that PMNL are in a primed statein both continuous ambulatory peritoneal dialysis (CAPD) andhemodialysis (HD) patients (7,8). In addition to CKD patients,we have shown PMNL priming as a common denominator in otherclinical states, such as hypertension, diabetes, and cigarettesmoking, that are known to be associated with endothelial dysfunction,accelerated atherosclerosis, and increased prevalence of cardiovascularmorbidity and mortality (9–11). In all of these clinicalstates, it was apparent that primed peripheral PMNL contributeconcomitantly to chronic systemic oxidative stress (OS) andinflammatory processes and that PMNL priming was associatedwith a significant increase in peripheral white blood cells(WBC) and PMNL counts, although still in the upper quadrantof the normal range (7–11). Recently, epidemiologic studieshave suggested that elevated WBC and neutrophil counts constitutea mortality predictor in HD patients (12,13) and are a riskfactor for developing CKD in U.S. adults (14). We suggest thatthe elevation in peripheral PMNL counts is a feature of systemiclow-grade inflammation derived from PMNL priming. Therefore,we designed a prospective, cross-sectional study aimed to characterizePMNL priming in relation to PMNL counts and the severity ofrenal failure in CKD patients before RRT was commenced and inpatients who are on CAPD and HD treatment.
Patients
A total of 120 participants, 90 CKD patients and 30 healthy,normal control subjects (NC), were enrolled in this cross-sectionalstudy after giving informed consent for blood sampling. Patientswith evidence of acute or chronic infection or malignancy orwho had received a blood transfusion within 3 mo before bloodsampling were excluded. This study was approved by the institutionalcommittee in accordance with the Helsinki declaration.
The patients were divided into three groups: No RRT, CAPD, andHD (Table 1):
30 stage 2 to 5 CKD patients who were not receivingRRT andhad estimated GFR ranging between 8 and 73 ml/min per1.73 m2;GFR was calculated according to the Modification ofDiet inRenal Disease (MDRD) formula (15).
30 CAPD patientswith mean duration of dialysis treatment of20 ± 4 mo(range 3 to 55 mo). All patients underwentdialysis with 8 L/din four exchanges (three isotonic 1.36%and one hypertonic 3.86%glucose solutions).
30 patients who were undergoing HD andhad a mean duration ofdialysis treatment of 47 ± 5 mo(range 9 to 90 mo). Allpatients underwent HD thrice weekly;each dialysis treatmentlasted 4 h and was carried out withlow-flux polysulfone membranes(F8; Fresenius Medical Care,Bad Homburg, Germany) using bicarbonatedialysate with an averagesingle pool Kt/V of 1.2 ± 0.2.The water for dialysismet the standards of the Associationfor the Advancement ofMedical Instrumentation.
Table 1. Clinical and biochemical characteristics of CKD patients and normal control subjectsa
PMNL and Sera Separation
Blood was drawn in the morning after an overnight fast fromall patients and NC for the determination of biochemical andhematologic parameters and for PMNL isolation. Blood from HDpatients was always drawn immediately before a dialysis session.PMNL isolation was carried out from a 20-ml heparinized bloodsample as described previously (10). The separated PMNL (>98%pure, approximately 107 cells per isolation) were resuspendedin PBS that contained 0.1% glucose. Sera were frozen at –20°Cfor C-reactive protein (CRP) quantification (Quantex, Biokit,Spain) using Hitachi 917 Automatic analyzer (Roche Diagnostics,Mannheim, Germany) and for IL-6 determination (Quantikine HS;R&D Systems, Minneapolis, MN).
PMNL-Mediated OS Rate of Superoxide Release.
The measurements of the rate of superoxide release are basedon superoxide dismutase inhibitable reduction of 80 µMcytochrome C (Sigma, St. Louis, MO) to its ferrous form (16).The rate of superoxide release was monitored from 106 separatedPMNL: (1) under resting conditions, at 22 and 37°C, forup to 90 min, without any stimulant; (2) after stimulation with0.32 x 10–7 M phorbol 12-myristate 13-acetate (PMA; Sigma),at 22 and 37°C for 50 min; and (3) after stimulation withzymosan (Sigma) at 37°C as described previously (10). Briefly,zymosan (4 mg; Sigma) was opsonized within 1 wk of the experiment,in 1 ml of pooled human sera of 10 healthy donors for 30 minat 37°C. This solution was washed twice with 154 mM NaCl,resuspended in 154 mM NaCl at a concentration of 10 mg/ml, andstored at –80°C until used. The particle-to-PMNL ratiowas 15. PMNL priming was determined by the rate of superoxidereleased from PMA-stimulated 106 PMNL at 22°C in 10 min.
PMNL Myeloperoxidase Activity.
The method is a combination of the methods of Suzuki et al.(17) and Bradley et al. (18), measuring myeloperoxidase (MPO)activity after solubilization of cell membrane. Briefly, eachfrozen pellet of 106 PMNL in 50 mM potassium phosphate (KPH)buffer was thawed and centrifuged at 20,000 x g at 4°C for20 min, then resuspended in 0.1 M KPH buffer (pH 6) that contained0.5% hexadecyltrimethyl ammonium bromide and 0.5 M EDTA andhomogenized. KPH buffer (80 mM; pH 5.4) that contained 0.5%hexadecyltrimethyl ammonium bromide and 16 mM 3,3',5,5'-tetramethylbenzidine was added to this suspension. The reaction was initiatedby adding 0.001% H2O2 and stopped at 30-s intervals for 90 sby adding 0.01 mg/ml catalase (specific activity 13,600 U/mgsolid; Sigma) at 4°C. The absorbance was detected at 650nm, and the activity was expressed as OD/min.
PMNL-Derived Inflammation WBC and PMNL Counts.
Counts of WBC and PMNL from blood drawn in EDTA were performedby an automated cell counter (Coulter STKS, Miami, FL) and usedas a measure of inflammation.
PMNL Survival Ex Vivo.
Separated PMNL, from 10 age- and gender-matched patients, fromeach group (10 CKD individuals, serum creatinine 3.7 ±1.04 mg/dl, range 2.1 to 5.4; 10 CAPD; 10 HD, and 10 NC), wereused at a concentration of 106 cell/ml. Duplicate samples ofPMNL were incubated with either autologous or heterologous pooledsera (25% vol/vol diluted with Hank’s balanced salt solution)at 37°C and counted before and after 90 min of incubation.Cell viability was confirmed by trypan blue (0.1% wt/vol) exclusion.PMNL survival was expressed as the ratio of cell counts after90 min of incubation to their counts before incubation (%).
Analysis of Apoptotic PMNL.
Apoptosis was analyzed in whole blood from 20 patients and controlsubjects of each group by flow cytometry according to Kuyperset al. (19). Blood samples were assayed for apoptosis afterlysis of red blood cells by Q prep (Beckman Coulter, Fullerton,California) and incubated with FITC-labeled mAb using the AnnexinV kit (Bender MedSystems, Vienna, Austria). PMNL were definedby forward scatter/side scatter and by R-phycoerythrin–labeledmonoclonal anti-CD16.
Statistical Analyses
Data are expressed as mean ± SEM. Differences in meanvalues were tested by two-way ANOVA and by the Bonferroni multiplecomparison test, using Prism version 3.0 statistical software(GraphPad Software, San Diego, CA). Correlations between differentstudy parameters were performed using Pearson correlation coefficients.P < 0.05 was considered significant.
Study Population Table 1 summarizes the clinical and biochemical characteristicsof the participants. All studied groups of patients showed similarmean values of BP, serum cholesterol, triglycerides, and glucose.Serum creatinine levels were increased as expected. Reducedserum albumin and increased serum CRP and IL-6 were found inall three renal failure groups of patients, as compared withNC, with significantly higher CRP levels in HD.
PMNL-Mediated OS Rate of Superoxide Release. Effect of Temperature.
The assay was performed at 22 and 37°C with resting or PMA-stimulated,separated PMNL (Figure 1). PMA stimulation caused an elevationin superoxide release in HD and in NC PMNL. After 10 min at22°C, the reduction of cytochrome C by superoxide releasedfrom PMA-stimulated HD PMNL was significantly faster than byNC PMNL. At 37°C, the two cell populations (HD and NC) releasedsuperoxide at much faster rates than at 22°C, and the significantdifference between these two cell populations was abolishedat 10 min. The rate of superoxide release was negligible inthe resting state compared with PMA-stimulated cells, for bothcell populations and temperatures studied. However, althoughNS, resting HD compared with resting NC PMNL showed a tendencyfor higher rates of superoxide release at both temperatures.Altogether, to emphasize the differences between HD and NC PMNL,all further superoxide release experiments were performed at22°C after PMA stimulation for 10 min (Figure 1).
Figure 1. Representative figure demonstrating the rates of superoxide release measured by superoxide dismutase (SOD)-inhibitable reduction of cytochrome C at 549 nm, from resting, nonstimulated, 106 separated polymorphonuclear leukocyte (PMNL) from normal control subjects (NC) and patients on hemodialysis (HD) at 22 and 37°C, and from NC and HD PMNL, after stimulation by 0.32 x 10–7 M phorbol 12-myristate 13-acetate (PMA), at 22 and 37°C. Rate of superoxide release is expressed as nmol/106 cells.
Effect of Stimulant.
Significantly faster rates of superoxide release from PMA-stimulatedPMNL were found in the three renal failure groups as comparedwith NC (Figure 2A), reflecting a higher priming state in allgroups versus NC. In PMNL from HD patients, the rate of superoxiderelease was highest and significantly higher than CKD (Figure 2A).For ruling out the effect of hypertension or diabetes onthe rate of superoxide release, intercomparison among threemain subgroups of CKD patients before RRT, according to theirunderlying diseases (diabetes, hypertension, and others), wasperformed (n = 10 in each subgroup). No significant differencesin the rates of superoxide release from PMNL of these subgroupswere observed (data not shown).
Figure 2. Rate of superoxide release by separated, PMA-stimulated 106 PMNL from NC, patients with chronic kidney disease (CKD), patients who are on continuous ambulatory peritoneal dialysis (CAPD), and patients who are on HD. Rate of superoxide release was measured by SOD-inhibitable reduction of cytochrome C, followed spectrophotometrically at 549 nm after stimulation of 106 separated PMNL by PMA (n = 30 in each group; A) or by zymosan (n = 20 in each group; B). Rate of superoxide release is expressed as nmol/106 cells per 10 min. Data are mean ± SEM. a,b,cP < 0.0001, NC versus CKD, CAPD, and HD patients, respectively; dP < 0.05, HD versus CKD; eP = 0.001, NC versus CKD, CAPD, and HD.
In contrast to PMA, challenging the same cell with zymosan showeda significant decrease in the rate of superoxide release inPMNL from each of the three groups of renal failure patientsas compared with NC (Figure 2B). No differences in zymosan-stimulatedsuperoxide release among the three renal failure groups (Figure 2B)could be demonstrated.
PMNL MPO Activity.
MPO activity from PMNL lysates of the three renal failure groupswas similar but significantly higher than MPO activity in NCPMNL lysate (Table 2).
Table 2. PMNL-derived oxidative and inflammatory markers of CKD patients and NCa
PMNL-Derived Inflammation WBC and PMNL Counts.
CKD, CAPD, and HD patients had significantly higher numbersof WBC and PMNL, as compared with NC (Table 2), although allvalues fell within the upper quartile of the normal range. WBCfrom CAPD and HD patients were significantly higher than thosefrom CKD patients. PMNL counts from HD patients were significantlyhigher than those from CKD patients.
Percentage of Apoptotic PMNL.
The percentage of apoptotic PMNL, assayed immediately afterblood withdrawal in whole blood, was significantly higher inall three groups of renal failure patients as compared withNC (Table 2).
PMNL Survival Ex Vivo.
PMNL that were isolated from peripheral blood of CKD, CAPD,and HD patients and from NC were incubated in their autologoussera for 90 min and counted by Coulter counter before and after90 min of incubation. Figure 3 shows that PMNL from CKD, CAPD,and HD patients exhibit a significant lower survival versusPMNL from NC, with the lowest significant survival of PMNL fromHD patients. Cross-incubation studies of cells from each groupwith NC sera were performed to clarify further whether the decreasedsurvival is influenced by humoral factors. NC sera significantlypromoted cell survival of PMNL from CKD, CAPD, and HD, withthe smallest recovery, although significant, in HD PMNL (Figure 3).
Figure 3. Survival of PMNL from NC, CKD, CAPD, and HD patients determined after 90 min of incubation in autologous () and (NC) heterologous () sera. PMNL survival was expressed as the ratio of cell counts after 90 min of incubation to their counts before incubation (%). Data are mean ± SEM; n = 10, for each experiment. aP < 0.05, PMNL from CKD patients versus NC; bP < 0.05, PMNL from CAPD patients versus NC; cP < 0.001, PMNL from HD patients versus NC; dP = 0.0006, PMNL from CKD patients incubated in autologous sera versus incubation in NC sera; eP = 0.002, PMNL from CAPD patients incubated in autologous sera versus incubation in NC sera; fP = 0.01, PMNL from HD patients incubated in autologous sera versus incubation in NC sera.
Relationship between PMNL Priming and Peripheral Counts
A positive significant correlation was found between the rateof superoxide released by 106 cells from each individual andPMNL counts in all participants (r = 0.33, P = 0.0002; n = 120;Figure 4).
Figure 4. Correlation between the rates of superoxide release from separated PMA-stimulated PMNL and peripheral PMNL counts. Data refer to PMNL from all renal failure patients and NC (n = 120).
PMNL Priming in Relation to GFR
PMNL priming expressed by the rate of superoxide release inNC and CKD patients not on RRT was negatively correlated withthe calculated GFR (by MDRD: r = –0.35, P = 0.0089; n= 60; Figure 5A); the lower the kidney function, the higherthe superoxide release. The peripheral PMNL counts from CKDpatients and NC were also negatively correlated with the valuesof calculated GFR (r = –0.55, P < 0.0001; n = 60; Figure 5B);the lower the GFR, the higher the number of PMNL.
Figure 5. PMNL priming related to the severity of kidney disease. (A) Correlation between the rates of superoxide release from separated PMA-stimulated PMNL and calculated GFR of NC and CKD patients not receiving renal replacement therapy (RRT; n = 60). (B) Correlation between peripheral PMNL counts and calculated GFR of NC and CKD patients not receiving RRT (n = 60).
Systemic Inflammatory Markers in Relation to GFR
Albumin, CRP, and IL-6, the widely accepted systemic inflammationmarkers, determined in NC and in CKD patients not on RRT werecorrelated with the calculated GFR (MDRD). Serum CRP levelsnegatively correlated with GFR (r = –0.44, P = 0.0077;n = 25; Figure 6A). IL-6 levels negatively correlated with GFR(r = –0.46, P = 0.011; n = 32; Figure 6A). Albumin levelspositively correlated with GFR (r = 0.72, P < 0.0001; n =60; Figure 6B).
Figure 6. C-reactive protein (CRP), IL-6, and albumin levels related to the severity of kidney disease. (A) Correlation between serum CRP or IL-6 levels and calculated GFR of NC and CKD patients not receiving RRT (n = 32 and 25, respectively). (B) Correlation between serum albumin levels and calculated GFR of NC and CKD patients not receiving RRT (n = 60).
Systemic Inflammatory Markers in Relation to PMNL Priming
No correlation could be found between the rates of superoxiderelease from separated PMNL and either serum CRP levels (r =0.07, P = 0.68) or IL-6 levels (r = 0.06, P = 0.71). No correlationcould be found between peripheral PMNL counts with either serumCRP levels (r = 0.0003, P = 1.0) or IL-6 levels (r = –0.13,P = 0.45). However, albumin levels negatively correlated withboth PMNL priming parameters: The rates of superoxide releasefrom separated PMNL (r = –0.22, P = 0.04) and with theperipheral PMNL counts (r = –0.26, P = 0.007).
The results of this study implicate PMNL priming in the causeof systemic OS and low-grade inflammation associated with renalfailure. The augmented superoxide release together with theincreased intracellular MPO activity are known contributorsto systemic OS. The extent of PMNL priming correlates positivelywith the severity of kidney disease and is intensified by RRT,especially by HD. The notion that the increased rate of superoxiderelease is related directly to the severity of renal failurein CKD patients who are not on RRT is supported by others (4).The likelihood that hypertension (9) or diabetes (10) per seis a contributor to PMNL priming in this study was ruled out,because no significant differences could be seen among the subgroupsof the enrolled CKD patients. Recently, Agarwal (20) reacheda similar conclusion, reporting that CKD is associated withOS independent of hypertension.
In our findings, the priming of PMNL from CAPD patients didnot differ significantly from CKD patients who were not on RRT,although Tarng and colleagues (21,22) observed increased PMNLpriming in CAPD patients. The significantly enhanced primingof HD PMNL versus CKD not on RRT occurred despite blood withdrawalbefore HD session and may reflect accumulating nondialyzed uremictoxins as well as possible accumulating side effects of theextracorporeal treatment (23–25). From this and otherstudies (6), it seems that uremia per se is a major contributorto PMNL priming: PMNL priming was almost twofold higher in CKDpatients who were not on RRT, compared with NC, whereas hemodialysisfurther increased the priming of PMNL by only 25%. The polysulfonelow-flux membrane used in this study is probably not the maincause of PMNL priming. Rao et al. (26) supported this notion,reporting that superoxide release from PMNL is similar for bothlow- and high-flux polysulfone membranes. Uremic toxins presentin the uremic milieu (6,22,27) are well-established factorsand may carry leukoclastic activity, as suggested by our exvivo cross-incubation studies.
The clear distinction among the different studied groups inPMNL priming was achieved by the in vitro use of lower assaytemperatures, 22 instead of 37°C. Thus, solely for the purposeof slowing the reactions to emphasize the differences betweenthe groups, we used nonphysiologic temperature, 22°C. Paulet al. (27) showed that the resting rates of superoxide releasedfrom both HD and NC PMNL were similar at 37°C. In our study,we have shown for the first time that at physiologic 37°C,the rate of superoxide release from resting HD PMNL is fasterthan NC, a phenomenon that is supported by the observationsat 22°C. These findings suggest that in HD patients, thevascular wall is chronically and continuously exposed to ROSgenerated from resting PMNL. ROS generated near the vascularwall, when improperly scavenged, may be the cause for endothelialdysfunction found in these patients.
Stimulation of PMNL with different stimulants resulted in different,even opposite, effects: PMA stimulation of PMNL caused a fasterrelease of superoxide, whereas stimulation with zymosan induceda slower release in all three renal failure groups, comparedwith NC. Zymosan, a physiologic stimulant that differs in mechanismof action compared with PMA, is used to assess the phagocyticpotential of PMNL. Although we did not look at phagocytosisdirectly, the slower rates of superoxide released extracellularly,compared with NC, suggests that the overall response to thisingested particle is reduced in PMNL from renal failure patients.The increased percentage of apoptotic PMNL in the circulationof renal failure patients demonstrated in this study, togetherwith the decreased phagocytic-like function, reflects a declinein innate immunity. Altogether, these observations can explain,at least in part, the reported high prevalence of infectiouscomplications, a major cause of morbidity and mortality, inthese patients (28,29). It should be emphasized that in thisstudy, the increased apoptosis of PMNL was determined immediatelyafter withdrawal in whole blood. Other studies have also shownincreased apoptosis in CKD patients; however, to the best ofour knowledge, the determination of apoptosis in the unmanipulatedsamples is novel and probably reflects best in vivo conditions,because these PMNL were not affected by either separation (30)or ex vivo long incubations (31).
OS, the well-documented observation in CKD patients before andwhile on RRT (4,6,20–25), can originate from PMNL priming,followed by chronic release of ROS and increased MPO activity(32). This study indicates that the activity of MPO is higherin PMNL that were obtained from all renal failure patients ascompared with NC. The increased MPO activity in primed PMNLin this study is similar to the MPO enrichment reported in primedmacrophages under inflammatory conditions (33). Augmented intracellularMPO activity, associated with chronic release of ROS and increaseddegranulation (34), can explain the higher plasma MPO activityreported by Chen et al. (35). Because MPO recently became apredictor of cardiovascular disease (36), the increased MPOactivity in PMNL from CKD patients may constitute a link betweencirculating PMNL and the risk for developing cardiovascularcomplications in these patients. PMNL priming, a source of chronicsuperoxide and MPO released near the vascular wall, may initiateand propagate the development of atherosclerosis, a common long-termcomplication of renal failure.
A new interesting correlation was observed between the ratesof superoxide released from 106 cells and peripheral PMNL numbers:The faster the rate, the more PMNL are found in the circulation.This increased PMNL number in the circulation is probably anadaptive response to superoxide chemoattraction (34) resultingin an elevation in peripheral PMNL counts. Hence, we proposethat PMNL priming can serve as a new measure of systemic low-gradeinflammation, involved in the deterioration of kidney function.This PMNL-mediated low-grade inflammation can explain the epidemiologicstudies showing increase in WBC counts as a mortality predictorin HD patients (12,13) and as a predictor for developing CKD(14).
We show that systemic inflammatory markers such as CRP, IL-6,and albumin correlate with GFR. The low-grade inflammation derivedfrom PMNL priming correlates significantly with GFR and albuminbut does not correlate with IL-6 and CRP. These interestingfindings suggest that different processes are involved in inflammation,which need to be clarified further.
In conclusion, our data suggest that PMNL priming is a key mediatorin inducing a vicious cycle of systemic OS and inflammationin CKD patients. The characteristics of the priming agents remainto be elucidated.
Acknowledgments
We thank Prof. J. Kopple for the critical review of the manuscript.The assistance of E. Ron and Dr. M. Furmanov is gratefully acknowledged.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication November 11, 2004.
Accepted for publication April 28, 2005.
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Abstract
Introduction
OD/min. 
) and (NC) heterologous (
) sera. PMNL survival was expressed as the ratio of cell counts after 90 min of incubation to their counts before incubation (%). Data are mean ± SEM; n = 10, for each experiment. aP < 0.05, PMNL from CKD patients versus NC; bP < 0.05, PMNL from CAPD patients versus NC; cP < 0.001, PMNL from HD patients versus NC; dP = 0.0006, PMNL from CKD patients incubated in autologous sera versus incubation in NC sera; eP = 0.002, PMNL from CAPD patients incubated in autologous sera versus incubation in NC sera; fP = 0.01, PMNL from HD patients incubated in autologous sera versus incubation in NC sera.
