renal transplant candidates present immune dysregulation , caused by chronic uremia followed by renal replacement therapy . recently ,
uremia was reported to influence cytokine synthesis . while the immune response shift is well described in the literature , still it is not clear whether the pretransplant chronic inflammation affects posttransplant outcome . from routine blood tests
crp ( c - reactive protein ) and albumin levels were shown not only to reflect the general immune response in uremic patients   but also to predict kidney outcome [ 4 , 5 ] .
pretransplant cytokine profiles were also considered to exert a deleterious effect on graft function or survival , but the published data have been inconclusive [ 6 , 7 ] .
the aim of the study was to investigate whether pretransplant peripheral blood gene expression affects clinical outcome of renal allograft recipients . in a prospective study we analyzed a wide range of immune factors known to be related to inflammation ( il-6 , il-8 , ngal , and tnf - alpha ) , apoptosis ( fas , caspase 3 , p53 , and il-18 ) , and lymphocyte t activation ( ifn - gamma , il-2 ) as well as regulatory t cell ( il-10 , tgf - beta , and foxp3 ) function with the real - time pcr method .
pretransplant cytokine gene expression linked to recipient- and donor - related factors was examined with further analysis of allograft function .
the working hypothesis was the search for the novel predictive biomarkers connecting the recipients ' pretransplant immune and inflammatory status with transplant outcome .
the study was carried out prospectively on 87 low risk consecutive renal transplant recipients ( aged 1672 years , mean 47 years ; 34 females , 53 males ) transplanted between 2006 and 2012 at wroclaw medical university . among them
they remained on renal replacement therapy from 1 to 97 months ( mean 25  18 months ) .
the patients received organs from donors ( 81 deceased , 6 living ) aged from 16 to 72 years .
eighty - three recipients received their first graft , while for 4 of them it was the second transplant .
immunosuppressive therapy consisted of corticosteroids with cyclosporine and mycophenolate mofetil / sodium in 35 cases , corticosteroids with tacrolimus and mycophenolate mofetil / sodium in 49 cases , corticosteroids with everolimus and tacrolimus in 1 case , and corticosteroids with sirolimus and cyclosporine in 2 cases .
four recipients received induction therapy with anti - cd25 antibodies at the time of transplantation .
pretransplant demographic and clinical data ( age , gender , weight , height , comorbidity , and history of chronic kidney disease ) were obtained from medical records from patients ' dialysis centers .
posttransplant clinical data ( renal function , immunosuppressive therapy , complications , and outcome ) were collected from medical records of the transplant outpatient clinic of the department of nephrology and transplantation medicine in wroclaw , poland .
estimated glomerular filtration rate ( egfr ) was calculated using the abbreviated modification of diet in renal disease study formula ( mdrd ) .
blood samples for routine laboratory tests assessing pretransplant inflammatory response as well as samples for gene expression were collected immediately before the transplant procedure and introduction of immunosuppressive therapy .
the study was approved by the commission of bioethics at wroclaw medical university , and all aspects of the study were in accordance with the world medical association declaration of helsinki . before enrollment , each patient read an information sheet and provided fully informed consent .
rna was isolated with a paxgene blood rna kit ( preanalytics ) and reversely transcribed with high capacity rna to cdna kit ( applied biosystems ) .
the peripheral blood gene expression of caspase-3 ( casp3 , hs00263337_m1 ) , fas ( fas , hs00236330_m1 ) , p53 ( tp53 , hs00153349_m1 ) , foxp3 ( foxp3 , hs00203958_m1 ) , ifn - gamma ( ifng , hs00174143_m1 ) , interleukin-2 ( il-2 , hs00174114_m1 ) , interleukin-6 ( il-6 , hs00174131_m1 ) , interleukin-8 ( il-8 , hs00174103_m1 ) , interleukin-10 ( il-10 , hs00174086_m1 ) , interleukin-17 ( il-17a , hs99999082_m1 ) , interleukin-18 ( il-18 , hs00155517_m1 ) , ngal ( lcn2 , hs00194353_m1 ) , tgf - beta ( tgfb1 , hs99999918_m1 ) , and tnf - alpha ( tnf , hs00174128_m1 ) was assessed with real - time pcr on custom - designed low density arrays ( taqman ) with taqman pcr master mix .
gapdh was chosen as the reference gene , and it was proved not to be related to transplant outcome ( delayed graft function ( dgf)/acute rejection occurrence ( ar ) , serum creatinine , and gfr ) .
each gene was tested in triplicate ; raw expression data were averaged and converted to ct , where ct = ctgene  ctgapdh and ct is the cycle threshold value and defines the calculated cycle number , in which the fluorescence measured during pcr reaction increases over the preset threshold value .
the expression data were analyzed separately for each of the genes in the subsequent manner .
the samples with the highest quartile of ct values ( which corresponds to the lowest expression level ) were chosen as a reference group ( ref ) for data normalization .
the expression data throughout the paper are presented as ct = mean ctref  ctsample .
statistical analysis was performed with the statistica v.12 statistical package ( statsoft , poland ) .
the numerical variables were tested for outliers and normality and presented as mean  standard deviation or median + interquartile range . in case of normally distributed variables ,
if the normality assumption was not fulfilled , mann - whitney u - test and spearman correlation were used .
for normally distributed continuous variables , their influence on the outcome was corrected by multiple regression for the other parameters shown significantly in univariate analysis .
delayed graft function ( defined as a need of hemodialysis in the first week after transplantation ) was seen in 20 cases ( 23% ) .
seventeen recipients ( 19% ) experienced at least one biopsy - proven rejection episode within the first posttransplant year .
recipients were followed up for 5 years after transplantation . during that time 5 recipients died ( 5.7% ) and 11 lost their grafts ( 12% ) .
severe cardiovascular complications occurred in 7 recipients ( 8% ) , serious infections with a need of hospitalization occurred in 10 cases ( 11% ) , 13 ( 15% ) recipients suffered from cmv disease , and 18 recipients ( 21% ) presented recurrent urinary tract infections .
after 24 months of observation 36% of recipients presented excellent kidney function with gfr above 50  ml / min ( mean gfr 60.3  5.4  ml / min ) .
recipients and donors characteristics are included in table 1 and the clinical pretransplant recipient blood parameters are presented in table 2 .
all the parameters were analyzed in relation to graft short- and long - term outcome .
the pretransplant blood parameters ( crp , albumin , cholesterol , complete blood cell test , etc . ) did not affect posttransplant outcome or acute rejection episodes .
moreover , in our study group , we found no association of cold ischemia time or number of hla mismatches and graft function or survival . from the clinical parameters studied only donor age , recipient age , and recipient bmi proved to influence allograft outcome . both recipients ' and donors ' age were negatively correlated with immediate and short- and long - term allograft function at any time point studied .
recipients ' age was associated with worse graft function from 14 days to 24 months ( 14-day gfr r = 0.33 , p = 0.003 ; 1-month gfr r = 0.37 , p = 0.001 ; 3-month gfr r = 0.31 , p = 0.0 ; 6-month gfr r = 0.39 , p = 0.001 ; 12-month gfr r = 0.33 , p = 0.008 ; and 24-month gfr r = 0.35 , p = 0.012 ) .
donor age was a strong risk factor for graft function deterioration from the beginning ( 14-day gfr r = 0.33 , p = 0.004 ; 1-month gfr r = 0.41 , p < 0.001 ; and 3-month gfr r = 0.44 , p < 0.001 ) up to 24 months ( 6-month gfr r = 0.60 , p < 0.001 ; 12-month gfr r = 0.48 , p < 0.001 ; and 24-month gfr r = 0.55 , p < 0.001 ) .
donor age was also a risk factor for delayed graft function ( 52.2  10.4 versus 42.8  13.8 ; p = 0.001 ) and acute rejection ( 52.1  15.0 versus 43.3  13.0 ; p = 0.018 ) .
recipients ' pretransplant bmi was negatively correlated with the short - term graft function from 3 up to 12 months ( 3-month gfr r = 0.35 , p = 0.009 ; 6-month gfr r = 0.36 , p = 0.010 ; and 12-month gfr rs = 0.30 , p = 0.033 ) .
the correlation between the strongest clinical factors as donor age and recipient bmi with short - term kidney function ( expressed by 6-month gfr ) is presented in figure 1 .
gene expression in the peripheral blood stabilized in paxblood rna tubes was studied with low density taqman arrays in triplicate .
pretransplant white blood cell ( wbc ) affected expression of foxp3 , il-2 , lcn2 , and tnf .
we observed the association of some gene expression with subsequent graft function ( table 5 ) . 

tgfb1 exerted a negative influence on short - term graft function ( 14-day gfr r = 0.24 , p = 0.039 ) .
pretransplant il-2 gene expression positively correlated with kidney function in the immediate posttransplant period ( 14-day gfr rs = 0.24 , p = 0.040 ) . 

lcn2 exerted a negative influence on short - term graft function ( 3-month gfr r = 0.27 , p = 0.024 ) . from th1-derived cytokines
, ifng was significantly upregulated in recipients with good kidney function at 6 months after ktx ( r = 0.27 , p = 0.029 ) . from apoptosis - related genes casp3 and fas
were positively correlated with short - term graft function ( casp3 : 6-month gfr r = 0.27 , p = 0.027 ; fas : 6-month gfr r = 0.28 , p = 0.021 ) . the long - term kidney function described by egfr from 12 months to 24 months
was positively correlated with pretransplant expression of proapoptotic genes casp3 and fas ( casp3 : 12-month gfr r = 0.3 , p = 0.016 , 24-month gfr r = 0.41 , p = 0.003 , and fas : 24-month gfr r = 0.3 , p = 0.033 ) .
60 months after transplantation casp3 and ifng association was still observed ( casp3 60-month gfr : rs = 0.37 , p = 0.037 ; ifng 60-month gfr : rs = 0.27 , p = 0.024 ) . also il-18 expression
correlated positively with 5-year graft function ( 60-month gfr rs = 0.39 , p = 0.024 ) .
the fourth examined proapoptotic gene , tp53 , did not correlate with graft function at any time point . from th1-derived cytokines , ifng was significantly downregulated in recipients with bad clinical outcome from 12 to 24 months after ktx ( 12-month gfr : r = 0.3 , p = 0.018 , 24-month gfr r = 0.29 , p = 0.04 ) .
also pretransplant il-2 gene expression positively correlated with kidney function in the late period ( 18-month gfr rs = 0.28 , p = 0.049 ; 60-month gfr rs = 0.41 , p = 0.026 , resp . ) .
we noted no association of expression of other examined genes with graft function at any time point .
no correlation was observed between gene profiles and delayed graft function or acute rejection episode rates .
as the particular time point gfr was influenced by a number of continuous variables , both clinical parameters ( donor age , recipient age , and recipient bmi ) and gene expression ( casp3 , fas , ifng , il-18 , tgfb1 , and lcn2 ) multiple regression tests were performed . for each time
point the parameters presenting significant correlation in simple regression analysis were included in multiple regression model as summarized in table 5 . from the clinical factors donor age was the strongest risk factor for graft function deterioration from the beginning up to 24 months .
also recipient age was proved to be significant up to 12 months , and an association with bmi was shown 6 months after ktx . 

tgfb1 gene expression was significantly and independently of donor and recipient age associated with the short - term allograft function ( 14 days ) .
ifng association with 6-month gfr was shown independently from donor age as well as recipient age and bmi .
the proapoptotic casp3 was shown to be associated positively with long - term graft function ( 12 and 24 months after ktx ) independently from donor age .
the impact of lcn2 seen after 3 months in univariate analysis was not confirmed in multiple regression .
we also studied whether any of the expression levels of the three candidate genes : casp3 , fas , and il-18 could predict better long - term allograft function ( egfr > 50  ml / min 24 months after ktx ) .
each of casp3 , fas , and il-18 gene expression levels above cut - off proved to have around 70% sensitivity and at least 80% specificity for good long - term outcome prediction .
we studied the immune background of renal transplant candidates just before the transplantation and its impact on the transplant outcome .
we analyzed the expression of immune - related genes at the mrna level with real - time pcr methodology .
we observed that th1 derived cytokines ( il-2 and ifn - gamma ) were significantly correlated with better short- and long - term allograft function .
we also observed a positive correlation between pretransplant expression of three proapoptotic genes , casp3 , fas , and il-18 , with allograft function . both of the observations were not published before .
in addition , it is possible to hypothesize that some level of th1 constitutive activation supports more active apoptosis which in turn is associated with secretion of anti - inflammatory , tolerogenic molecules as il-10 and tgf - beta .
collectively , these observations could be interpretative as a credit for hypothesis that some basal immune system cell activation is needed to make them more sensitive to effective inhibition by immunosuppressive drugs .
it was caused by abnormal function of regulatory t - cells , the depletion of dendritic cells , naive and central memory t cells and b cells , and impaired phagocytic function of polymorphonuclear cells and monocytes [ 1 , 8 , 9 ] .
various studies have shown that pretransplant immune status of the recipient can influence transplant outcome , acute rejection episodes , delayed graft function , and kidney survival .
it was noted that pretransplant humoral autoimmunity ( cross matches , panel reactive antibodies ) affected graft outcome .
previously , we as well as other authors have reported that cellular alloimmunity measured by the elispot technique influenced the acute rejection rate and overall graft survival [ 11 , 12 ] . in our population
some studies observed that pretransplant blood cytokine pattern reflected immune infiltrates observed in kidney tissue during rejection .
similarly to some previous reports , we observed that none of the pretransplant cytokine patterns was associated with ar .
th1 cytokines ( il-2 , ifn - gamma ) trigger an alloimmune response with activation of t cytotoxic lymphocytes , monocytes , and nk cells .
pretransplant levels of anti - inflammatory cytokine tgf - beta tended to be lower among patients with acute rejection episodes , in contrast to upregulated th1-derived cytokines .
pretransplant serum levels of another proinflammatory cytokine , il-6 , were associated with an increased risk of acute rejection episodes and graft loss , and sil-6r was higher and tgf - beta was lower in patients exhibiting atn .
unexpectedly , it was noted that pretransplant elevations of not only proinflammatory il-12 , but also anti - inflammatory cytokine il-10 , were both connected to acute rejection .
in contrast there are few reports of no relationship of pretransplant cytokine levels with acute rejection incidence .
chin et al . observed that pretransplant ifn - gamma levels did not predict acute rejection in a small retrospective study .
. found no significant difference in serum concentration of il-2 , il-10 , il-4 , and ifn - gamma on the day before transplantation between the group with acute rejection within the first months and rejection - free recipients .
did not observe any influence of pretransplant levels of examined cytokines il-1 , il-2 , il-3 , il-4 , il-6 , il-10 , tnf - alpha , tgf - beta , and ifn - gamma on acute rejection or atn rate . among other conflicting reports we observed no correlation between immune factors ' gene expression and acute rejection .
nevertheless in our study pretransplant gene expression of th1-derived cytokines ( il-2 and ifng ) significantly correlated with better posttransplant allograft function from the beginning up to 5 years . according to our knowledge
, no previous papers have reported an association between pretransplant cytokine expression and kidney allograft function measured by gfr .
th1-derived cytokines trigger an alloimmune response but also are easily suppressed by standard immunosuppressive drugs ( calcineurin inhibitors act by inhibiting t cells through il-2 deprivation ) .
furthermore , it is known that ifn - gamma inhibits synthesis of the potent rejection - maintaining cytokine il-17 and further inhibits th2 differentiation .
this is a pathway of switching the alloresponse to a cellular rather than a humoral reaction .
the humoral mechanism of rejection was recently described as more harmful and more difficult to manage than the cellular type .
graft failure has been described rarely after t - cell - mediated rejection and commonly after antibody - mediated rejection . moreover ,
some authors have observed elevated levels of ifn - gamma in stable patients after renal transplantation compared to an acute rejection group [ 13 , 19 ] .
that mechanism can partially explain our observation that higher pretransplant ifn - gamma expression was linked to better short- and long - term allograft function .
we observed for the first time in the literature a positive correlation between pretransplant expression of three proapoptotic genes : for caspase-3 , fas , and il-18 ( casp3 , il-18 , and fas ) , with allograft function expressed as egfr .
that association was seen not only in the early posttransplant period , but also in the long - term observation up to 5 years .
increased apoptosis of leukocytes was reported to be associated with increased proinflammatory activity in patients with chronic kidney disease .
fas , which is an apoptosis triggering cytokine , increased significantly with the stage of chronic kidney disease , especially in patients treated with peritoneal dialysis .
we did not find any published reports concerning expression of apoptosis - related genes in relation to further allograft function . in our study
pretransplant expression of tp53 , which belongs to the major apoptosis - inducing gene family , correlated negatively with wbc count .
the discovered positive correlation between proapoptotic gene expression and graft function may be partially explained by increased apoptosis of neutrophils and lymphocytes , which are known to be involved in the graft injury . from the standard markers of inflammation , pretransplant crp
was reported to be a risk factor for acute rejection episodes   or not to influence graft function and survival among children   or adults . in the present study we also found no influence of pretransplant crp levels on the rate of delayed graft function or acute rejection and allograft function .
after transplantation the restoration of renal function reduces the intensity of chronic inflammation in allograft recipients .
severe pretransplant hypoalbuminemia was reported to be an independent risk factor for delayed graft function or graft loss [ 4 , 26 ] .
in contrast , we did not find higher risk of posttransplant dgf , ar , or worse graft function in hypoalbuminemic recipients , possibly because the majority of our recipients presented pretransplant albumin levels within the normal range .
we noted that the albumin level correlated significantly with il-10 gene expression in our patients .
this is an expected finding because hypoalbuminemia was described as a potent factor augmenting chronic inflammation [ 2729 ] . in our study recipients ' pretransplant bmi
contrary results were published on the association of a higher 1-year gfr with pretransplant obesity and lower early gfr in patients with bmi < 18.5  kg / m .
more conflicting results on severe obesity and kidney allograft function were reported [ 31 , 32 ] .
found that recipient bmi correlated with the incidence of dgf and 1- and 5-year allograft function .
we did not find recipients ' bmi to be a risk factor for acute rejection or dgf , probably because of low prevalence of obese and undernourished patients in our population ( mean bmi was around 24  kg / m ) . among donor - related issues , age is one of the most important factors negatively influencing outcomes of kidney allografts .
like other authors , we found a strong correlation between donor age and kidney function from the beginning up to 2 years .
apart from nonspecific time - related injury , kidneys from older donors were proved to be associated with a more intense host immune response early after transplant ( up to 1 month ) reflected by significantly higher numbers of peripheral t and b cells , increased t - cell alloreactivity , and modified cytokine patterns in rats .
allografts from older donors were reported to be more immunogenic than those from younger donors [ 36 , 37 ] . in our study ,
donor age was an independent significant risk factor not only for delayed graft function but also for acute rejection , which is in agreement with previous reports .
this deleterious influence was still noticeable despite the fact that the majority of our recipients ( 86% ) received grafts from donors younger than 60 years .
all the previously published results on the predictive value of pretransplant cytokine measurements were based on determination of cytokine blood concentration by elisa technique .
our report for the first time assesses the cytokine gene expression in relation to posttransplant outcome .
it was previously reported in a murine model that the ifn - gamma gene expression detected by real - time kinetic rt - pcr correlated very well with ifn - gamma protein secretion measured by elisa .
examining serum levels of cytokines in kidney allograft recipients together with blood mrna expression did not find any significant differences in those two ways of assessment of cytokine production .
the study was performed to investigate whether pretransplant peripheral blood immune gene expression affects clinical outcome of renal allograft .
the lowered pretransplant th1-derived cytokine and apoptosis - related gene expressions were a hallmark of subsequent worse kidney function but not of acute rejection rate .
the pretransplant ifng and casp3 and fas and il-18 genes expression in the recipients ' peripheral blood is the possible candidate for novel biomarker of short- and long - term allograft function .
 renal transplant candidates present immune dysregulation , caused by chronic uremia . 
 the aim of the study was to investigate whether pretransplant peripheral blood gene expression of immune factors affects clinical outcome of renal allograft recipients . 
 methods . in a prospective study , we analyzed pretransplant peripheral blood gene expression in87 renal transplant candidates with real - time pcr on custom - designed low density arrays ( taqman ) . results . 
 immediate posttransplant graft function ( 14-day gfr ) was influenced negatively by tgfb1 ( p = 0.039 ) and positively by il-2 gene expression ( p = 0.040 ) . 
 pretransplant blood mrna expression of apoptosis - related genes ( casp3 , fas , and il-18 ) and th1-derived cytokine gene ifng correlated positively with short- ( 6-month gfr casp3 : p = 0.027 , fas : p = 0.021 , and ifng : p = 0.029 ) and long - term graft function ( 24-month gfr casp3 : p = 0.003 , fas : p = 0.033 , il-18 : p = 0.044 , and ifng : p = 0.04 ) . 
 conclusion . 
 lowered pretransplant th1-derived cytokine and apoptosis - related gene expressions were a hallmark of subsequent worse kidney function but not of acute rejection rate . 
 the pretransplant ifng and casp3 and fas and il-18 genes ' expression in the recipients ' peripheral blood is the possible candidate for novel biomarker of short- and long - term allograft function . 
1. Introduction
2. Patients
3. Methods
4. Results
5. Discussion
6. Conclusion