brain - derived neurotrophic factor ( bdnf ) is a member of the nerve growth factor family and it exerts its effects by binding to the tropomyosin kinase b receptor . among the neurotrophins
bdnf is a basic dimeric protein , and its subunit is a 13.5 kda ( 119 amino acids ) molecule .
it is an essential mediator of neuronal activity and synaptic plasticity in both the central and peripheral nervous systems , and it enhances the growth and maintenance of several neuronal systems by serving as a neurotransmitter modulator 1 .
it is well known that the bdnf expression increases following an insult to the central nervous system ( cns ) .
although many studies have demonstrated a relevant role of bdnf for regulating neuroprotection , recent findings have suggested that changes of plasma bdnf level , regardless of nervous system , are also associated with several inflammatory or metabolic derangement conditions 2 - 4 .
decrease of renal function induces accumulations of circulating cytokines , advanced glycation end product and pro - oxidants , which contributes to the pro - inflammatory milieu in patients with chronic kidney disease .
uremic inflammation is associated with various adverse outcomes in hemodialysis ( hd ) patients , such as cardiovascular disease , malnutrition and neurological disorders .
while many studies have investigated inflammatory cytokines as a reflection of uremic condition in hd patients , there is no information about the peripheral changes of bdnf in patients undergoing hd as far as we are aware . in this cross - sectional study
, we investigated the changes of the plasma bdnf levels in hd patients and evaluated the potential correlations between this peptide and selected parameters of uremic inflammation .
using a cross - sectional design , we studied 60 randomly selected , stable uremic patients who were undergoing hd for more than 6 months and 30 healthy control subjects who were matched for age and sex .
patients with known hypersensitivity to the dialysis membrane , signs of infection , malignancy , severe liver disease , autoimmune disease , previous history of renal transplantation , treatment with drugs known to interfere with the immune system were excluded . following an overnight fast ,
blood samples were collected from both hd patients and control subjects . for the hd patients
, the samples were obtained from the arterial side at the time of needling before the dialysis session .
fasting blood was withdrawn into a lithium heparin vacuum tube and immediately centrifuged at 3,800 rpm for 10 minutes .
human plasma bdnf was assayed using the duoset elisa development system ( catalog number : dy248 , r&d systems , uk ) .
all the assays were performed in duplicate using the manufacturer 's recommended buffers , diluents and substrates .
the intra - assay and inter - assay coefficients of variation were 5.4% and 9.8% , respectively .
the concentrations of the samples in each plate were calculated according to each standard curve and the dilution factor .
the cytokine assays for human serum il-2 , il-4 , tumor necrosis factor- ( tnf- ) and interferon- ( ifn- ) were performed using the highly sensitive hs - elisa development system from r&d systems , abingdon , uk .
the intra- and inter - assay coefficients of variation were 5.6 and 7.08% , respectively , for il-2 , il-4 , and tnf-. the intra- and inter - assay coefficients of variation were 6.7 and 9.2% for ifn- 5 .
the presence of diabetes , body mass index ( bmi ) , dialysis duration and dialysis adequacy ( kt / vurea ) were assessed in the hd patients .
we also measured hemoglobin , hematocrit , platelet count , blood urea nitrogen , creatinine , serum albumin , lipid profiles , calcium , phosphorus , intact parathyroid hormone ( ipth ) , high sensitivity c - reactive protein ( hs - crp , normal value < 3.0 mg / l ) , ferritin and fibrinogen in these patients .
the data with continuous variables and a normal distribution were presented as means  standard error , and those without a normal distribution were presented as the median with ranges .
the kolmogorov - smirnov test showed that the bdnf values did not fit a standard distribution curve .
therefore , statistical analyses were performed using an appropriate non - parametric test , the mann - whitney u - test .
the relationship between bdnf levels and other clinical or biochemical parameters was also analyzed by multiple regression analysis .
using a cross - sectional design , we studied 60 randomly selected , stable uremic patients who were undergoing hd for more than 6 months and 30 healthy control subjects who were matched for age and sex .
patients with known hypersensitivity to the dialysis membrane , signs of infection , malignancy , severe liver disease , autoimmune disease , previous history of renal transplantation , treatment with drugs known to interfere with the immune system were excluded .
following an overnight fast , blood samples were collected from both hd patients and control subjects . for the hd patients
, the samples were obtained from the arterial side at the time of needling before the dialysis session .
fasting blood was withdrawn into a lithium heparin vacuum tube and immediately centrifuged at 3,800 rpm for 10 minutes .
human plasma bdnf was assayed using the duoset elisa development system ( catalog number : dy248 , r&d systems , uk ) .
all the assays were performed in duplicate using the manufacturer 's recommended buffers , diluents and substrates .
the intra - assay and inter - assay coefficients of variation were 5.4% and 9.8% , respectively .
the concentrations of the samples in each plate were calculated according to each standard curve and the dilution factor .
the cytokine assays for human serum il-2 , il-4 , tumor necrosis factor- ( tnf- ) and interferon- ( ifn- ) were performed using the highly sensitive hs - elisa development system from r&d systems , abingdon , uk .
all the assays were performed according to the manufacturer 's instruction . the intra- and inter - assay coefficients of variation were 5.6 and 7.08% , respectively , for il-2 , il-4 , and tnf-. the intra- and inter - assay coefficients of variation were 6.7 and 9.2% for ifn- 5 .
the presence of diabetes , body mass index ( bmi ) , dialysis duration and dialysis adequacy ( kt / vurea ) were assessed in the hd patients .
we also measured hemoglobin , hematocrit , platelet count , blood urea nitrogen , creatinine , serum albumin , lipid profiles , calcium , phosphorus , intact parathyroid hormone ( ipth ) , high sensitivity c - reactive protein ( hs - crp , normal value < 3.0 mg / l ) , ferritin and fibrinogen in these patients .
the data with continuous variables and a normal distribution were presented as means  standard error , and those without a normal distribution were presented as the median with ranges .
the kolmogorov - smirnov test showed that the bdnf values did not fit a standard distribution curve . therefore
, statistical analyses were performed using an appropriate non - parametric test , the mann - whitney u - test .
the relationship between bdnf levels and other clinical or biochemical parameters was also analyzed by multiple regression analysis .
the plasma bdnf level was significantly higher in hd patients than in control subjects ( median 630.3 , range 76.4 - 1435.0 vs. 312.4 , 78.7 - 969.9 pg / ml , respectively , p < 0.05 ) ( figure 1 ) .
serum il-2 , il-4 , tnf- and ifn- also showed higher values in the hd group than in the control group ( p < 0.05 ) .
there were no significant differences in age , gender and bmi between the hd group and the control group ( table 1 ) .
the baseline characteristics of the hd patients are summarized in table 2 . among the 60 hd patients , 26 ( 43.3% ) had diabetes .
the median duration of dialysis was 43.5 months ( range , 7 - 240 months ) .
we classified hd patients into subgroups according to age , gender and the presence of diabetes , and compared the plasma bdnf levels between subgroups .
there were no significant differences in the plasma bdnf level in subgroup analysis according to age (  60 years , median 592.7 , range 90.5 - 1380.0 pg / ml ;  60 years , median 635.7 , range 76.4 - 1435.0 pg / ml ; p = 0.83 ) and gender ( male , median 512.0 , range 76.4 - 1435.0 pg / ml ; female , median 625.3 , range 90.5 - 1412.6 pg / ml ; p = 0.40 ) .
however , the plasma bdnf level of diabetic hd patients showed higher value compared to those of non - diabetic hd patients and control subjects ( diabetic hd patients , median 822.4 , range 90.5 - 1435.0 pg / ml ; non - diabetic patients , median 418.7 , range 102.4 - 835.0 pg / ml ; control subjects , median , 312.4 , range 78.7 - 969.9 pg / ml ; p < 0.01 ) .
furthermore , non - diabetic hd patients showed slightly , but significantly higher plasma bdnf value than control subjects ( p = 0.022 ; figure 2 ) .
correlation analysis was performed to examine the relationships between logbdnf and the clinical or biochemical parameters in the hd group .
the logbdnf was positively correlated with hs - crp ( r = 0.409 , p < 0.01 ) and ifn- ( r = 0.551 , p < 0.01 ) , and it was negatively correlated with serum albumin ( r = -0.408 , p < 0.01 ) .
in multivariate regression analysis , logbdnf was independently related to the presence of diabetes ( -coefficient = 0.399 , p = 0.041 ) and ifn- ( -coefficient = 0.538 , p = 0.003 ) after adjustment for age , sex , bmi , dialysis duration , serum albumin , crp and cytokines ( table 3 ) .
according to many other studies , bdnf has been shown to protect the cns under a variety of conditions .
previous work demonstrated that patients with fibromyalgia and depression have increased serum bdnf concentrations 6 .
it has also been shown that plasma bdnf concentrations were elevated in drug users or in patients with chronic stress 7,8 . recently ,
apart from nervous systems , there are several reports showing the associations of plasma bdnf with systemic or peripheral inflammatory conditions , such as diabetes , acute coronary syndrome , atherosclerosis or rheumatoid arthritis 2 - 4 . in this study , we investigated for the first time the changes of the plasma bdnf level in uremic condition and the correlations between bdnf and clinical or biochemical parameters in hd patients .
we found that the plasma bdnf level was significantly elevated in patients who were undergoing hd compared to the control subjects .
the plasma bdnf was removed during dialysis session . in our study , the average plasma bdnf clearance of a single hd session , which is defined as the pre - hd blood levels minus the post - hd blood levels divided by the pre - hd blood levels , was ~ 19.4% , and the median post - hd plasma bdnf level was 508.1 pg / ml ( range , 25.1 - 1327.0 pg / ml ) .
the post - hd plasma bdnf level was significantly higher than that of the controls ( p < 0.05 ) . however , there was no correlation between the post - hd plasma bdnf and the clinical or biochemical parameters including cytokines ( data not shown ) . in the present study ,
the increase of the bdnf to be more marked in the hd patients with diabetes .
previous animal research demonstrated that subcutaneous administration of bdnf reduced food intake and ameliorated impaired glucose tolerance in diet - induced obesity mice 9 .
furthermore , systemic bdnf treatment normalized fasting blood glucose , hemoglobin a1c and lipid profiles 10,11 .
therefore , in the present study , it is possible that plasma bdnf may increase in hd patients with diabetes to compensate hyperinsulinemia and insulin resistance .
however , there is a controversy as to whether the serum bdnf level is higher in diabetics than in non - diabetics who are not undergoing dialysis treatment .
a previous study showed that serum bdnf level was higher in newly diagnosed type 2 diabetic patients than in normal controls 2 . on the other hand ,
fujinami et al . reported that serum bdnf levels were significantly lower in patients with advanced type 2 diabetes compared to control subjects 13 . in our results , most of diabetes in dialysis group were not newly diagnosed , and might be more complicated diabetes .
therefore , the presence of diabetes alone could not explain the elevated level of the plasma bdnf in the present study .
our results also showed that the plasma bdnf level in non - diabetic hd patients was higher than that of control subjects .
there may be many other factors including uremic inflammation to increase the plasma bdnf level in diabetes of the hd patients .
there were no differences of plasma bdnf level according to age or gender subgroups in our hd patients .
conflicting results exist as regards the changes of bdnf associated with age or gender differences .
a recent report showed that a significant age - related increase of the plasma bdnf level in down 's syndrome patients 14 .
however , there are also reports showing age - related decrease of the serum bdnf level or no significant difference of the serum bdnf in age 6,13 . in the gender aspect ,
a previous report showed that bdnf levels were significantly higher in females than in males with type 2 diabetes 2 .
in contrast , laske et al . revealed no significant gender difference of the serum bdnf in fibromyalgia patients 6 .
it seems that gender or age - related serum bdnf results could differ according to underlying disease and study population .
recent studies demonstrated that pro - inflammatory cytokines , such as il-1 , il-6 , tnf- and ifn- showed higher value .
anti - inflammatory cytokine levels including il-2 , il-4 , il-5 and il-12 were decreased in dialysis patients 15 . in the present study ,
in addition , the hd patients showed a significantly positive relationship between the plasma bdnf and the serum ifn- in multivariate regression analysis .
increases of bdnf expression in inflamed bladder tissues and in airway epithelium during allergic airway inflammation have been demonstrated 16,17 .
furthermore , grimsholm et al reported that plasma bdnf level in rheumatoid arthritis patients showed higher values than in healthy controls , and anti - tnf treatment lead to a decrease in the plasma bdnf level 4 .
it is unclear that higher plasma bdnf in the hd patients was associated with uremic inflammation .
it has been shown that activated antigen - specific t cells , b cells , and monocytes produced bdnf 18 .
uremic inflammatory state is characterized by enhanced immune system which involves both t lymphocytes and monocytes 19 .
therefore , it is postulated that chronic inflammatory condition and enhanced immune system is associated with the production of bdnf , which may explain the reason that the plasma bdnf level in non - diabetic hd patients was higher compared to that of control subjects in this study .
serum contains platelets , and it has been well established that large amount of bdnf is stored in platelets 20 . it was previously reported that serum bdnf level was found to be 200-fold higher than plasma level 21 .
thrombin , collagen , the ca ionophore a23187 and shear stress stimulated a rapid release of bdnf from platelets 20 .
thus , the difference between serum and plasma levels of bdnf could explain the amount of bdnf stored in platelets , and plasma bdnf may reflect circulating levels rather than the levels stored in platelets 22 .
only a few data are available on plasma bdnf levels and it is difficult to determine the significance of bdnf of plasma according to disease state .
nofuji et al . recently reported that the serum bdnf concentration was found to significantly change according to exercise , however , the plasma bdnf level was not 23 . on the other hand , piccinni et al .
showed in their study with depressed patients that while the plasma bdnf levels increased in parallel with the clinical improvement after one month antidepressant treatment , they could not detect any significant change in serum bdnf levels in spite of the treatment 24 . in the future
, studies will be required to determine the action and regulation of plasma bdnf in peripheral blood according to disease state .
first , this is a cross sectional study , therefore , longitudinal observation is needed to assess the relationship between this peptide with uremic inflammatory conditions and clinical outcomes .
second , this study is limited by its small sample size . further study with a large dialysis population would add further information about the role of bdnf as markers of inflammation and malnutrition including hs - crp and serum albumin .
third , in this study , we measured only the plasma levels of bdnf , and this was the major limitation of the study .
the source of plasma bdnf and its biological activity were not investigated in the present study .
future studies are needed to determine which source of bdnf - platelets , plasma , serum , or whole blood - provides the most reliable biological marker of patients with hd .
finally , we did not provide data on the assessment of nervous system functions in the patients and the other neurotrophins such as nerve growth factor or neurotrophin-3 .
the evaluation of these parameters in the hd patients may help to explain the change of the plasma bdnf level in uremic condition . in conclusion ,
the plasma bdnf level showed higher values in the hd patients , and the presence of diabetes seemed to be associated with the higher value of the plasma bdnf in the patients .
in addition , the plasma bdnf level was found to be positively correlated with inflammatory cytokine in these patients , which suggests that plasma bdnf might reflect the uremic inflammatory condition in the patients undergoing hd .
 objective : brain - derived neurotrophic factor ( bdnf ) is a neurotrophin which modulates neuronal survival and proliferation . 
 recently , plasma bdnf is associated with inflammatory conditions . 
 the present study was to investigate the changes of the plasma bdnf level in hemodialysis patients and to evaluate the relationship between the plasma bdnf and uremic inflammation.method : we measured the plasma bdnf , serum il-2 , il-4 , ifn- and tnf- in hemodialysis patients and control subjects . 
 in addition , we investigated the associations of the plasma bdnf with clinical or biochemical parameters.results : the plasma bdnf level was significantly higher in hemodialysis patients than in the control subjects ( median , 312.3 vs. 630.3 pg / ml , p < 0.05 ) . in subgroup analysis , hemodialysis patients with diabetes showed higher values of the plasma bdnf than the patients without diabetes . 
 however , there were no significant differences in age and gender subgroups of the hemodialysis patients . 
 the logbdnf was positively correlated with hs - crp and ifn- , and negatively correlated with serum albumin . in multivariate regression analysis , the logbdnf was independently associated with the presence of diabetes ( -coefficient = 0.399 , p = 0.041 ) and ifn- ( -coefficient = 0.538 , p = 0.003).conclusion : the plasma bdnf may increase in hemodialysis patients , and more prominently in the patients with diabetes . 
 furthermore , the plasma bdnf might reflect inflammatory condition in hemodialysis patients . 