Positive correlation of serum leptin with blood lymphocytes in maintenance hemodialysis patients

Vol. 5, No. 4, 2005 • The Journal of Applied Research524

Positive Correlation of SerumLeptin with Blood Lymphocytes inMaintenance Hemodialysis PatientsHamid Nasri, MD*Azar Baradaran, MD†

* Nephrology Research Center (Hemodialysis Department), Hajar Medical, Educational andTherapeutic Center, Shahrekord University of Medical SciencesShahrekord, Iran†Department of Biochemistry, Center of Research and Reference Laboratory of IranHospital Bu Ali, Tehran, Iran

hemodialysis adequacy, and a significantinverse correlation of white blood cellcounts with serum albumin were seen.

Conclusion: Generally, increased neu-trophil counts and reduced lymphocytecounts are independent predictors ofincreased mortality risk in hemodialysispatients. Although this study and othersshowed positive association of serumleptin with lymphocytes and an inversecorrelation of serum leptin with PMNs,the authors conclude a protective rolefor leptin in decreasing mortality inhemodialysis patients; therefore, serumleptin in hemodialysis has a reverse epi-demiology role for maintaining immunesystem function in hemodialysis.

INTRODUCTIONPatients on chronic hemodialysis sufferfrom general immune incompetence.1

Malnutrition as a cause of immuneincompetence in dialysis patients is acommon clinical problem in patientswith end-stage renal disease (ESRD)and is generally the result of poor foodintake.2-3 Malnutrition is an independentfactor causing morbidity and mortality.4Leptin is an adipocyte-secreted hor-mone that centrally regulates weightcontrol.5 However, leptin receptor is

KEY WO R D S : l e p t i n , l y m p h o cy t e d s,h e m o d i a l y s i s

ABSTRACTObjective: The objective of this studywas to find the association of serum lep-tin with blood lymphocyte and polymor-phonuclear (PMN) percentages as themarkers of immune system function aswell as nutritional status in maintenancehemodialysis patients.

Design: In a group of maintenancehemodialysis patients, serum leptin,albumin, creatinine, blood urea nitrogen,and white blood cell counts consisting oflymphocytes and PMN cells were meas-ured.

Results: A significant positive correla-tion of serum leptin with body massindex, a significant positive correlationof serum leptin with lymphocyte per-centage, and a significant inverse corre-lation of serum leptin with PMNpercentage were seen. Near significantinverse correlations of white blood cellcounts with duration and dosage of dial-ysis, a near significant inverse correla-tion of white blood cell counts with

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expressed not only in the central nerv-ous system, but also in other systemssuch as hematopoietic tissues.

Human leptin has previously beenshown to enhance cytokine productionby murine peritoneal macrophages andhuman circulating monocytes.6 Leptinbelongs to the helical cytokine familyand its plasma concentrations correlatewith fat mass and respond to changes inenergy balance. Initially, leptin was con-sidered an anti-obesity hormone, butexperimental evidence has shownpleiotropic effects of this molecule onhematopoiesis, angiogenesis, lymphoidorgan homeostasis, and T-lymphocytefunctions. More specifically, leptin linksthe pro-inflammatory T-helper (Th)-1immune response to nutritional statusand energy balance. Indeed, decreasedleptin concentrations during conditionsof food deprivation lead to impairedimmune capabilities.7 Malnutrition andthe consequent reduction of the fat masscause immunodeficiency in animals andhumans.8-9 Reports have recently shownthat leptin deficiency is responsible forthe immunosuppression and the thymicatrophy observed during acute starva-tion and undernutrition.10-11

After nutritional deprivation, leptinblood levels fall because of reductions inbody fat, causing impairment of theimmune function. This effect also hasbeen demonstrated in animals distant inthe evolutionary scale such as insects.12

Therefore, leptin seems to be one of themajor players in the immunoendocrinescenario, regulating the correlationamong nutritional status, basal metabo-lism, and immune function.10

Furthermore, the presence of leptinis necessary for an effective cell-mediat-ed immune response.10 Indeed, CD4+ T-lymphocyte activities are suboptimal inthe absence of leptin.10 Serum leptin lev-els are elevated in patients with ESRDand in hemodialysis, and experimentalevidence suggests a possible role for lep-tin in the development of protein-energymalnutrition in this population.13-14

Indeed, in the last few years, we havedeveloped an increasing understandingof how immunosuppression occurs dur-ing malnutrition.

The focus has shifted from nutrientsto hormones, in particular leptin. Leptininduces oxidative stress in humanendothelial cells. It is possible that highleptin levels in renal failure may further

Figure 1: Near significant difference of white blood cell (WBC) counts between males andfemale hemodialysis (HD) patients.



enhance renal oxidative stress.15 Releaseof leptin from adipocytes may be stimu-lated by cytokines mediating the inflam-matory response, which is frequentlypronounced in patients with ESRDreceiving hemodialysis.14-16 Oxidativestress and an increased total white bloodcell (WBC) count has been found tocorrelate with increased cardiovascularmortality in elderly men.17 An associa-tion between WBC counts and mortalityin ESRD also has been suggested.17 Tofind the association of serum leptin withblood lymphocyte and PMN percentageas the markers of immune system func-tion as well as nutritional status andwhile a high WBC and a low percentageof lymphocytes are associated with a sig-nificant increase in mortality and hospi-talization in maintenance hemodialysispatients,18 we therefore aimed to con-duct a study on ESRD patients underregular hemodialysis to explore thisassociation.

PATIENTS AND METHODSThis cross-sectional study was conductedon patients with ESRD who wereundergoing maintenance hemodialysis

treatment with acetate-based dialysateand polysulfone membranes. Accordingto the severity of secondary hyper-parathyroidism, each patient being treat-ed for secondary hyperparathyroidismwas given oral active vitamin D3(Calcitriol; Rocaltrol) (Roche Hexagon;Roche Laboratories Inc , New Jersey),calcium carbonate capsule, and Rena-Gel (sevelamer; Genzyme Europe B.V.;United Kingdom/Ireland) tablet at vari-ous doses. According to the severity ofanemia, patients were prescribed intra-venous iron therapy with Iron Sucrose(Venofer; Vifor (international) Inc.St. Gallen, Switzerland) at various dosesafter each dialysis session. All patientsreceived treatments of 6 mg folic aciddaily, 500 mg Acetyl- L-Carnitine(JarrowFormulas, Inc, Los Angeles, CA) daily,oral vitamin B-complex tablets daily,and 2,000 U intravenous Eprex (recom-binant human erythropoietin [Rhuepo]Janssen-Cilag; CILAG- AGInternational 6300 Zug, Switzerland)after each dialysis session.

Exclusion criteria were active orchronic infection and using drugs hadadverse effects on bone marrow.

Figure 2: Significant positive correlation of serum leptin and body mass index



Complete blood counts containing whiteblood cells (WBCs/uL) with lymphocyteand PMN cells differentiation weremeasured using Sysmex-KX-21N cellcounter (SYSMEX CORPORATION;Mundelein, Illinois, Sysmex America,Inc.). Levels of serum predialysis creati-nine and post- and predialysis BUNwere measured using standard kits.Serum leptin (normal range of valuesfor men is 3.84 ng/mL [±1.79] and forwomen is 7.36 ng/mL [±3.73]) was meas-ured by enzyme-linked immunosorbentassay (ELISA) using DRG kits (DRGDiagnostics, Berlin, Germany). The bodymass index (BMI) was calculated usingthe standard formula (postdialyzedweight in kg/m2).19 For the efficacy ofhemodialysis, the urea reduction rate(URR) was calculated from pre- andpost-BUN data.20

Duration and dosages of hemodialy-sis treatment were calculated from thepatients’ records. The duration of eachhemodialysis session was 4 hours.Statistical analysis was performed ontotal hemodialysis (HD), female, male,

diabetic, and nondiabetic populationsseparately. For statistical analysis, thedata are expressed as the mean ± stan-dard deviation (SD). Comparisonbetween the groups was done usingStudent’s t-test. Statistical correlationswere assessed using partial correlationtest. All statistical analyses were per-formed using SPSS software (version11.5.00) (SPSS Inc,Chicago,IL).Statistical significance was determinedat a P value <0.05.

RESULTSThere was a total of 39 patients (15women, 24 men), consisting of 27 nondi-abetic HD patients (11 women, 16 men)and 12 diabetic HD patients (4 women,8 men). Table 1 shows the patients’mean ± SD age, the length of time theywere on hemodialysis, the dialysisdosage, and the results of laboratorytests. The mean patient age was 46 years(±18).

The value of serum leptin of totalpatients was 10 ng/mL (±14) (median 6.8ng/mL). The percentage of lymphocytes

Figure 3: Significant positive correlation of serum leptin with lymphocyte percentage.



in total patients was 27% (±9) (median25%). In all patients no significant dif-ferences of lymphocyte and PMN per-centage and serum leptin between menand women were found. However, anear significant difference of WBCcounts between men and women wasseen (P=0.092; Figure 1). No significantdifferences of WBC counts, lymphocyte,PMN percentage, and serum leptinbetween diabetic and nondiabetic HDpatients were seen.

A significant positive correlation ofserum leptin with BMI (r=0.55; P<0.001;Figure 2) was seen. Although in totalpatients there was a significant positivecorrelation of serum leptin with lympho-cyte percentage (r=0.38; P=0.016; Figure3), only in nondiabetic HD patients werefound a significant inverse correlation ofserum leptin with PMN percentage (r=-0.48; P=0.013; Figure 4). In all patients,no significant correlations of WBCcounts, lymphocyte, and PMN percent-age with BMI were seen. In all patients,a near significant and inverse correlationof WBC counts with duration of

hemodialysis (r=-0.27; P=0.094) and infemale population a near significant andinverse correlation of WBC counts withhemodialysis dosage were seen (r=-0.52;P=0.055).

In all patients a near significant andinverse correlation of WBC counts withhemodialysis adequacy as determined byURR (r=-0.29; P=0.072) was seen(adjusted for age for all correlations).The association of dialysis adequacy (byURR) with WBC counts in the maleHD group was positively significant(r=0.49; P=0.017; Figure 5) (adjusted fordialysis dosage).

DISCUSSIONIn this study we found a near significantdifference of WBC counts between maleand female dialysis patients with morevalues among men. There was a signifi-cant positive correlation of serum leptinwith BMI, a significant positive correla-tion of serum leptin with lymphocytepercentage, and a significant inverse cor-relation of serum leptin with PMN per-centage. We also found a near significant

Figure 4: Significant inverse correlation of serum leptin with PMN percentage.



inverse correlation of WBC counts withduration of hemodialysis, a near signifi-cant and inverse correlation of WBCcounts with hemodialysis dosage, and anear significant and inverse correlationof WBC counts with hemodialysis ade-quacy.

Leptin was found to be in direct pro-portion to the amount and percentage ofbody fat.2 1 Leptin and the leptin receptorare part of a novel pathway that stimu-lates haemopoiesis.2 1 Leptin signalsthrough leptin receptor (Ob-R), which isa member of a class I cytokine receptorf a m i l y.2 2 It has been reported that Ob-Ris expressed also in human CD34+ cellsand that leptin administration inducesproliferation of human and murine stem-cells in vitro.2 3 Consistent with our find-ing in HD patients, Mabuchi andc o l l e a g u e s, endeavoring to find the rela-tion of WBC count and serum leptinc o n c e n t r a t i o n s, conducted a study with1,082 men and 200 women aged 40 to 59years (normal subjects) and showed asignificant and independent associationof WBC count and serum leptin concen-

t r a t i o n .2 4 In this regard, in a populationof 44,114 ESRD patients receivingh e m o d i a l y s i s, Reddan and colleagues2 5

found the following:• higher lymphocyte count was asso-

ciated with higher serum albumin andcreatinine,

• high neutrophil count was associ-ated with lower serum albumin and cre-atinine,

• increased lymphocyte count wasassociated with reduced mortality risk,and

• increased neutrophil count wasassociated with increased mortality risk.

In addition, it was shown that anincreased neutrophil counts is stronglyassociated with, and reduced lymphocytecounts associated less strongly with,many surrogates of both malnutritionand inflammation. An increased neu-trophil count and reduced lymphocytecount are independent predictors ofincreased mortality risk in hemodialysispatients.25

In our study, we also demonstratedthe negative association of leptin with

Figure 5: Significant positive correlation of dialysis adequacy by urea reduction rate (URR) withWBC counts .



Table 1. Minimum and maximum of age, duration and dosage hemodialysis, and laboratoryresults of hemodialysis patients

Total patientsN=39 Minimum Maximum Mean±SD MedianAge years 16 80 46±18 42DH* months 2 156 31±35 18Dialysisdose (sessions) 36 1584 279±381 156URR % 39 76 58.7±8.7 58BMI kg/m2 16 34 21.7±4.4 21Leptin ng/mL 0.10 73 10±14 6.8Creat mg/dL 3 18 9.5±3.6 9BUN mg/dL 30 180 83±33 76WBC counts/ uL 3000 11200 4860±2710 5500Lymphocyte % 11 47 27.2±8.9 25PMN % 40 85 65.3±11 67Non-diabeticHD patientsn=27 Minimum Maximum Mean±SD MedianAge years 16 80 42±17 49DH* months 2 156 37±40 21Dialysisdose (sessions) 36 1584 347±442 156URR % 50 76 60±7.6 60BMI kg/m2 16 33 21±4.4 19Leptin ng/mL 0.10 52 8±10 4Creat mg/dL 4 15 9.8±3 10BUN mg/dL 30 180 82±32 74WBC counts/ uL 3000 10300 5600±1800 5500Lymphocyte % 11 47 27±9 25PMN % 40 85 64.8±11.5 65Diabetic HD patients n=12 Minimum Maximum Mean±SD MedianAge years 27 79 55±17 57DH* months 6 24 15±6 15Dialysisdose (sessions) 54 216 127±59 132URR % 39 75 54±9.5 54BMI kg/m2 20 34 23±3.7 23Leptin ng/mL 0.20 73 14.4±19.5 9.4Creat mg/dL 3 17 8.7±4.7 9BUN mg/dL 30 140 85±35 90WBC counts /uL 3500 11200 4860±2710 6200Lymphocyte % 15 43 51±18 26.5PMN % 42 77 66.5±10.8 70

*Duration of hemodialysis treatment



PMN percentage. Negative associationsof WBC counts with duration and dosesof dialysis are the result of further toxicand suppressive effects of uremic toxinson bone marrow with increasing theduration of dialysis. Also, a positive cor-relation of WBC counts with dialysisefficacy may show that an adequate dial-ysis acts toward the resorting of ahematopoetic system.

As was explained regarding theassociation of nutritional deprivationand leptin levels in subjects withoutrenal failure, leptin blood levels fallbecause of a reduction in body fat, caus-ing impairment of the immune func-tion.8-12 It also has been described inhumans that leptin deficiency causesincreased frequency of and mortality byinfections early in life.26 In conditions ofnutritional deficit and reduced energystores, leptin plays a crucial function thatcontributes to induce all the adaptivemechanisms necessary to save energy,ensuring the correct function of vitalorgans such as the heart, kidney, andbrain.27 Indeed, the organism throughleptin deficiency reduces activation andexpansion of immune cells to sustainmore life-necessary functions.

Conversely, in conditions of excessand unbalanced leptin signals, togetherwith genetic, gender, and environmentalfactors, leptin can favor the break ofself-tolerance and at least in some ani-mal models can sustain and promoteCD4+ T-cell-mediated autoimmune dis-eases.28 To answer whether leptin plays arole in the immunosuppression of mal-nutrition in humans, Palacio and col-leagues studied children withprotein-calorie malnutrition, who hadslightly decreased fat and significantlylower leptin levels.29 This study showedthat there was no correlation betweenbody fat and leptin, reflecting the acutesuppressive effects of malnutrition. Withfeeding, leptin levels rapidly returned tonormal, with restoration of the correla-

tion to body fat before restoration ofnormal fat content. Likewise with feed-ing, the ability of the infants’ polymor-phonuclear white cells to makeinterferon-g and TNF-a increased,whereas production of IL-4 decreased.These changes are similar, but not iden-tical with what is found with leptin treat-ment of ob/ob(obese (ob) mouse strain )and starved mice.29

High leptin levels signal the pres-ence of sufficient energy stores to sitesin the central nervous system, whichrespond by reducing appetite andincreasing energy expenditure, prevent-ing severe obesity.30 Therefore, leptinsignals the nutritional status from theperiphery to the area of the braininvolved in the homeostasis of energybalance.30

The increased levels of leptin inhemodialysis patients are not only theresult of retention of the hormone, butprobably from increased production.Anorexia of hemodialysis patients hasbeen attributed to the increased leptinlevels, even if this is largely ahypothesis.31 We saw that in subjectswithout renal failure, malnutrition wasassociated with leptin deficiency. In con-trast to normal subjects, in hemodialysispatients malnutrition was associatedwith hyperleptinemia, which may be theresult of microinflamation status causedby hemodialysis treatment and theinflammatory stimuli, which have previ-ously been shown to induce elevatedsystemic leptin concentrations, propos-ing that leptin induction is part of theubiquitous acute phase reaction. Thishas been explained by the cytokineproperties of leptin and its receptor, asthe secondary structure of leptin resem-bles that of cytokines and the leptinreceptor is homologous to the signal-transducing subunit of the IL-6 receptorfamily.32-34

We showed a significant positivecorrelation of serum leptin with BMI.



More recent studies in maintenancedialysis patients suggest a paradoxicallyinverse association between higherserum leptin and improved markers ofnutritional status.33-34 This finding is con-sistent with the theory of reverse epi-demiology for leptin.33-34 Indeed, leptinhas been reported to be a negative acutephase reactant in ESRD patients.30 Thus,while an increased neutrophil count andreduced lymphocyte count are inde-pendent predictors of increased mortali-ty risk in hemodialysis patients, andwhile our study and others showed posi-tive association of serum leptin withlymphocytes, and inverse correlation ofserum leptin with PMNs, we can con-clude a protective role for leptin indecreasing mortality in hemodialysispatients and therefore serum leptin inhemodialysis has a reverse epidemiologyrole for maintaining the immune systemin hemodialysis.

ACKNOWLEDGMENTWe wish to thank Dr. K. Kalantar-Zadeh, Assistant Professor of Medicineof UCLA School of Medicine, for send-ing his articles to our center and for hiscomments.

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Positive correlation of serum leptin with blood lymphocytes in maintenance hemodialysis patients

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