The authors have declared that no competing interests exist.
Polymorphonuclear leucocytes are the first line of defence against foreign invaders and constitute the major cell type involved in certain types of acute and chronic inflammatory diseases.
The aim of the present study was to investigate the changes in expression of BCL-2 and BAK genes by real time PCR and to study whether they were involved in the accelerated neutrophil apoptosis which might be responsible for the recurrent bacterial infections seen in chronic renal disease and hemodialysis patients. Subjects and Methods: This study was conducted on sixty two subjects. Patients were selected from those admitted to Theodor Bilharz Research Institute (TBRI). Patients under study were classified into three groups; CKD patients (group I) kept on conservative treatment (22 cases), ESRD patients (group II) maintained on dialysis therapy, HD (20 cases). In addition, twenty healthy individuals served as a control group (group III) were involved. Results: There was significant increase in level of BAK gene in both patients' groups compared to control group with more increase in CKD group than ESRD group. Significant difference between the 3 groups was encountered with a higher expression level in CKD and ESRD groups than controls. There was decrease in level of BCL2 gene in both groups less than control group with more declines in ESRD group than CKD group.
Bcl2 and Bak genes could have a role in survival and apoptosis of the studied groups and suggested their impact in controlling the inflammatory mechanisms and eventually their therapeutic potential.
Polymorph nuclear leucocytes PMNLs are the first line of defense against foreign invaders and constitute the major cell type involved in certain types of acute and chronic inflammatory diseases
Impaired function of apoptotic neutrophils was suggested as a disorder; results in high frequency of bacterial infections that are the main cause of hospitalization and mortality among haemodialysis patients
The BCL-2 family contains pro- and anti-apoptotic proteins that are found in the outer membranes of mitochondria. The balance between the pro- and anti-apoptotic members of this family is the central factor in the opening and closing of permeability transition pores
The present work was aimed to investigate the changes in gene expressions of proapoptotic BAK and antiapoptotic BCL-2 genes; members of the BCL-2 family which may be involved in the accelerated neutrophil apoptosis and hence recurrent bacterial infections seen in CKD and hemodialysis patients and to clarify their potential therapeutic benefit.
Sixty two participants were enrolled in this work. Selected patients were classified into 3 groups; group I included 22 patients suffering from chronic kidney disease (CKD) on conservative treatment with no previous history of dialysis, they composed of 13 females (59%) and 9 males (41%) with a mean age of 51.72±12.70. Group II included 20 end stage renal disease (ESRD) patients maintained on hemodialysis programs 3 sessions weekly; 4h each; for a period of ≥3 months, they were represented by 5 females (25%) and 15 males (75%) with a mean age of 56.41±11.62. In addition to group III encompasses 20 healthy individuals serving as control group, they were represented by 9 males (45%) and 11 females (55%) with a mean age value 28.90 ± 6.83. The procedures used in this study were approved by Theodor Bilharz Research Institute (TBRI) ethics committee according to Helsinki Declaration. The two patient groups were selected from the Nephrology Medicine department, Theodor Bilharz Research Institute (TBRI). The normal control group was recruited from the hospital staff. This study was conducted during the period from October 2013 to April 2014.
All participants were subjected to detailed history taking attributed mainly to bacterial infection anywhere; the onset of the disease to determine whether it is of acute or chronic onset, the underlying etiology of renal failure. Thorough clinical examination to exclude patients who have splenomegaly, hepatomegaly, jaundice, other signs of hepatic failure or any sign of autoimmune disease and laboratory investigations including complete blood count, kidney function tests, liver function tests, hepatitis markers (HBs Ag & HCV Ab) and special laboratory investigations (for patients and controls) for detection of hSC-reactive protein in the serum by ELISA technique and neutrophil apoptosis genes (Bcl2 and BAK) by real time polymerase chain reaction technique according to the method described by Livak.1999
Ten milliliters of venous blood were collected from each patient and each individual of the control group by sterile venipuncture under complete aseptic conditions and divided as follows: 2 ml of venous blood on ethylene diamine tetra-acetic acid (EDTA) sterile vacutainer for performing complete blood picture, 5 ml on plain sterile vacutainer for performing KFT, LFT, hepatitis markers and hSCRP and 3 ml on EDTA for the study of neutrophil apoptosis genes (Bcl2 and BAK) by real time polymerase chain reaction technique.
Detection of HSC-reactive protein in serum with DRG diagnostic enzyme-linked immunosorbent assay (ELISA) as previously described.
Study of neutrophil apoptosis genes (Bcl-2 and BAK) by real time polymerase chain reaction technique
I. Neutrophil isolation
Leucocyte rich plasma was separated on a gradient of percoll after dextran sedimentation of RBCs. Percoll has a higher density than that of erythrocytes and granulocytes. Diluted blood is layered onto the density gradient (percoll). During the centrifugation process which follows, erythrocytes and granulocytes settle above the density gradient on account of their lower density
II. RNA isolation from neutrophils by using GeneJET Whole Blood RNA Purification Kit
III. Reverse transcription (Complementary DNA synthesis) by using Maxima First Strand cDNA synthesis kit
IV. Real time quantitative PCR
QuantiFast Probe Assays are predesigned assays based on hydrolysis probe detection (also known as TaqMan® assays). Each QuantiFast Probe Assay consists of a premixed primer pair and hydrolysis probe. Two dye labels are available: FAM™ (for all genes) and MAX™ (for selected reference genes). All QuantiFast Probe Assays are labeled with IBFQ (Iowa Black™ fluorescent quencher). IBFQ is a dark quencher with a broad absorbance spectrum ranging from 420 to 620 nm.
Kit for BAK gene assay (cat.no:QF00531048)
Kit for BCL2 gene assay (cat.noQF00301434)
The gene sequences for BAK, BCL-2, and ACTB were from the NCBI database (
Relative mRNA expression levels of BCL-2 gene (cat.noQF00301434) and BAK gene (cat.no:QF00531048) and ACTB gene (cat.no: QF00531209) were determined according to the 2-ΔΔCT method which asses fold change protein ratio calculated as follow: Calculation of the mean and standard deviation values of the sample results then we calculated the ΔCT value by ΔCT = CT target – CT reference. After that we calculated the standard deviation of the ΔCT value and the ΔΔCT value by ΔΔCT = ΔCT test sample – ΔCT calibrator sample. Then we calculated the standard deviation of the ΔΔCT value. Finally we incorporating the standard deviation of the ΔΔCT values into the fold-difference with 2–ΔΔCT
Results for subjects’ demographic and clinical characteristics are given as means ± standard deviations (SD) for continuous variables and n (%) for categorical variables. Results for mRNA expression and apoptosis percentages are given as means ±SD and median. For continuous variables, groups were compared using a two-sample t-test or a Mann–Whitney U test if data were not normally distributed. A Pearson chi-square test was used for categorical variables. Comparison between more than two groups was done by ANOVA test. Statistical significance was set at P < 0.05. Statistical analyses used SPSS 15.0 software.
Oral and written informed consent was obtained from all patients according to Helsinki guidelines of research ethics.
The patient's and the control groups demographic, clinical and laboratory characteristics are summarized in
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29-7251.72±12.70 | 26-7056.41±11.62 | 21-4128.90 ± 6.83 | |
9 (41%)13 (59%) | 15 (75 %)5 (25 %) | 9(45%)11(55%) | |
10 (45 %)12 (55 %) | 9 (45 %)11 (55 %) | 0 (0 %)20 (100%) | |
14 (64 %)8 (36 %) | 10 (50 %)10 (50 %) | 0 (0 %)20 (100%) | |
4 (18 %)18 (82 %) | 9 (45 %)11 (55 %) | 0 (0 %)20 (100%) | |
1 (5 %)21 (95 %) | 3 (15 %)17 (85 %) | 0 (0 %)20 (100%) | |
6.20 – 14.209.65 ± 1.89 | 6.00 – 12.009.45 ± 1.80 | 12.00 - 15.7012.94 ± 1.29 | |
3.20-10.207.18± 2.00 | 1.70-8.806.23± 1.67 | 4.40 – 8.206.69 ± 1.11 | |
62.00-800.00283.68 ± 134.24 | 104.00 – 339.00216.35 ± 68.09 | 150.00 – 390.00236.09 ± 57.89 | |
33.00-300.00140.54 ± 71.48 | 80.00 – 171.00118.65 ± 23.90 | 15.00 – 36.0023.27 ± 6.69 | |
1.65-12.006.28 ± 2.79 | 1.20 – 9.065.81 ± 2.49 | 0.40 –1.000.67 ± 0.19 | |
0.80- 32.606.50 | 0.80 – 9.403.60 | 0.78 – 5.202.07 |
Hb: Hemoglobin ; TLC: Total leucocytic count; HSCRP: highly sensitive CRP.
Expression levels of BAK and BCL-2 genes in CKD and ESRD groups showed an increase in expression level of BAK gene in both groups with higher level in CKD group than ESRD group. Median and mean values of BAK relative quantification (RQ) in CKD group were 1.17 and 2.01 respectively while median and mean values of RQ in ESRD group were 0.29 and 1.17 respectively. Level of BCL-2 shows low expression in both groups of patients with more declines in ESRD group than CKD group. Median and mean values of BCL-2 RQ in CKD group were 0.86 and 1.15 respectively and median and mean values of RQ in ESRD group were 0.32 and 0.9 respectively. Comparison of expression levels of BAK and BCL-2 genes in the 2 studied patient groups (
Item | CKD group(No; 22) | ESRD group(No; 20) | P value |
BAK RQ | |||
Range | 0.02 – 10.18 | 0.07 – 4.10 | |
Median | 1.17 | 0.29 | 0.04 |
Mean±SD | 2.01±2.2 | 1.17±1.51 | S |
BCL2 RQ | |||
Range | 0.04 – 3.61 | 0.02 – 3.42 | 0.1 |
Median | 0.86 | 0.32 | NS |
Mean±SD | 1.15±0.96 | 0.90±1.12 |
Comparison between CKD group and ESRD group subjects as regards expression levels of BAK and BCL-2 genes revealed a statistically significant difference between the two groups with a higher expression level of BAK gene in CKD than ESRD group (P= 0.04).
There was no statistically significant difference between the two groups regarding expression level of BCL-2 gene however a lower expression level of BCL-2 was elicited in ESRD than CKD group (P= 0.1).
Comparison of expression levels of BAK and BCL-2 genes in the 3 studied groups are shown in
Item | CKD group | ESRD group | Control group | P value |
(No; 22) | (No; 20) | (No; 20) | ||
BAK RQ | ||||
· Range | 0.02 – 10.18 | 0.07 – 4.10 | - | 0.05 |
· Median | 1.17 | 0.29 | 1 | S |
· Mean ± SD | 2.01±2.2 | 1.17±1.51 | 1.00±0.00 | |
BCL2 RQ | ||||
· Range | 0.04 – 3.61 | 0.02 – 3.42 | - | 1 |
· Median | 0.86 | 0.32 | 1 | NS |
· Mean ± SD | 1.15±0.96 | 0.90±1.12 | 1.00±0.00 |
: Significant;
: Non significant,
: relative quantitation
Comparison between CKD group, ESRD group subjects and controls as regards expression levels of BAK and BCL-2 genes revealed a marginally statistically significant difference between the 3 groups with a highest expression level of BAK gene in CKD than other two groups (P= 0.05).
There was no statistically significant difference between the 3 groups regarding expression level of BCL-2 gene however the lowest expression level was noticed among ESRD group than other two groups (P=1.00).
Comparison between CKD, ESRD and control groups as regards demographic and laboratory data (
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29-7251.72±12.70 | 26-7056.41±11.62 | 21-4128.90 ± 6.83 | 0.001 |
||
9 (41%)13 (59%) | 15 (75 %)5 (25 %) | 9(45%)11(55%) | 0.001 |
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6.20 – 14.209.65 ± 1.89 | 6.00 – 12.009.45 ± 1.80 | 12.00 - 15.7012.94 ± 1.29 | 1.20 |
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3.20-10.207.18± 2.00 | 1.70-8.806.23± 1.67 | 4.40 – 8.206.69 ± 1.11 | 1.25 |
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62.00-800.00283.68 ± 134.24 | 104.00 – 339.00216.35 ± 68.09 | 150.00 – 390.00236.09 ± 57.89 | 0.08 |
||
33.00-300.00140.54 ± 71.48 | 80.00 – 171.00118.65 ± 23.90 | 15.00 – 36.0023.27 ± 6.69 | 0.001 |
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1.20 – 9.065.81 ± 2.49 | 1.65-12.006.28 ± 2.79 | 0.40 –1.000.67 ± 0.19 | 0.001 |
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0.80- 32.606.50 | 0.80 – 9.403.60 | 0.78 – 5.202.07 | 0.001 |
: Highly significant;
: Non significant
Comparison between CKD group, ESRD group and control group subjects as regards clinical and laboratory data revealed statistically significant difference between the three groups concerning age and sex distribution and urea, creatinine and HSCRP levels with more prevalent age and males distribution among ESRD than other 2 groups and highest levels of urea, creatinine and HSCRP in CKD group than other 2 groups (P=0.001). However non-significant changes between the three groups according to Hb level, TLC and platelet count were shown.
Comparison between ESRD and CKD groups as regards clinical and laboratory data (
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29-7251.72±12.70 | 26-7056.41±11.62 | 0.22 |
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9 (41%)13 (59%) | 15 (75 %)5 (25 %) | 0.001 |
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10 (45 %)12 (55 %) | 9 (45 %)11 (55 %) | 1.00 |
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14 (64 %)8 (36 %) | 10 (50 %)10 (50 %) | 0.53 |
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4 (18 %)18 (82 %) | 9 (45 %)11 (55 %) | 0.09 |
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1 (5 %)21 (95 %) | 3 (15 %)17 (85 %) | 0.33 |
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6.20 – 14.209.65 ± 1.89 | 6.00 – 12.009.45 ± 1.80 | 0.72 |
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3.20-10.207.18± 2.00 | 1.70-8.806.23± 1.67 | 0.1 |
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62.00-800.00283.68 ± 134.24 | 104.00 – 339.00216.35 ± 68.09 | 0.06 |
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33.00-300.00140.54 ± 71.48 | 80.00 – 171.00118.65 ± 23.90 | 0.19 |
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1.65-12.006.28 ± 2.79 | 1.20 – 9.065.81 ± 2.49 | 0.56 |
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0.80- 32.606.50 | 0.80 – 9.403.60 | 0.001 |
: Highly significant;
: Non significant
Comparison between ESRD group and CKD group subjects as regards clinical and laboratory data revealed a highly statistical significant difference regarding sex distribution and HSCRP level. There was more prevalent females distribution and a higher level of HSCRP in CKD group (P=0.001) meanwhile there was no significant changes between the 2 groups in other clinical and laboratory data.
PMNL apoptosis is an important mechanism regulating the extent of an inflammatory response, rendering it self-limiting thus preventing excessive tissue damage. At sites of inflammation, complex interplay between proapoptotic and survival signals must control the outcome of the response. Therefore, it is important to prevent a reduced immune function in the case of increased apoptotic cell death or a state of increased inflammation in the case of reduced PMNL apoptosis
Furthermore, it has been noticed that immune system dysregulation in uremic patients may be accentuated by dialysis. In fact, during dialysis treatment cytokines and many other soluble uremic toxins may be partly responsible for a variety of functional disturbances that contribute to an increased risk of infection by interfering with essential functions of the non specific immune response such as chemotaxis, phagocytosis and oxidative metabolism
The aim of this study was to investigate the changes in gene expressions of proapoptotic BAK and antiapoptotic BCL-2 genes; members of the BCL-2 family which may be involved in the accelerated neutrophil apoptosis which may play a role in the recurrent bacterial infections seen in CKD and hemodialysis patients and to clarify their potential therapeutic benefit.
The study was conducted on 62 participants classified into 3 groups; group I included CKD patients kept on conservative treatment with no previous history of dialysis (22 cases), group II included ESRD patients maintained on haemodialysis therapy (20 cases), both groups were selected from those admitted to the Nephrology Departments and Hemodialysis Unit, Theodor Bilharz Research Institute (TBRI). In addition, group III included healthy individuals served as a control group. All individuals enrolled in the study were subjected to the following routine investigations; complete haemogram, kidney function tests, liver function tests and hepatitis markers HBs Ag, HCV-Abs, HIV Abs. In addition, inflammatory state was evaluated by estimation of CRP using HSCRP ELISA technique. Basically, this work was undertaken to study changes in relative gene expression of BCL-2 and BAK genes in neutrophil by using real time quantitative PCR technique (RQ-PCR).RQ-PCR system is based on the detection and quantification of fluorescent reporter. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template
In our study we utilized QuantiFast Probe Assays which are predesigned assays based on hydrolysis probe detection (also known as TaqMan® assays). Each QuantiFast Probe Assay consists of a premixed primer pair and hydrolysis probe. Two dye labels are available: FAM™ (for all genes) and MAX™ (for selected reference genes). All QuantiFast Probe Assays are labeled with IBFQ. IBFQ is a dark quencher with a broad absorbance spectrum ranging from 420 to 620 nm.
Detection of relative expression of BAK and BCL-2 genes were done using 2–ΔΔCT method which asses fold change protein ratio mentioned previously in methodology.
The present study revealed a statistically significant increase in the mean level of BAK gene (pro-apoptotic gene) in CKD group than in ESRD group (median values 1.17, 0.29 and mean values 2.01±2.2, 1.17±1.51 respectively) (P=0.04). Furthermore a marginally statistically significant difference between all patients groups (CKD, ESRD) and control groups was encountered with a higher mean expression level in CKD and ESRD groups than in controls (median values 1.17, 0.29 and 1 respectively) and (mean values 2.01±2.2, 1.17±1.51, and 1±0.0 respectively) (P= 0.05).These data means that BAK gene might play a role in acceleration of neutrophil apoptosis in uremic patients and consequently increases liability to recurrent bacterial infections known to occur among those patients.
In contrary to our findings Zhang et al.
Our study revealed also that there was decrease in the expression level of BCL-2 gene (anti-apoptotic gene) in both groups of patients compared to the control group with more decline in ESRD group than CKD group (median value 0.32, 0.86,1 respectively and mean values1.15±0.96,0.90±1.12respectively).These difference were not statistically significant between CKD group and ESRD group (P= 0.1); as well as between the three studied groups and each other (P= 0.1).These data mean that BCL-2 gene may play role in neutrophil apoptosis in ureamic patients and its decline help in acceleration of neutrophil apoptosis. These findings are matched with those of Majewska et al.
In contrast with the results of the present work concerning BCL-2 expression, Friedrich et al.
In agreement with the results of this work, Crokera et al.
Dounousi et al.
Data obtained from the present work regarding increase in expression level of BAK gene which is proapoptotic gene were in accordance with those of Zahran et al.
Musiał & Zwolin´ska
Our study was in agreement with those of Preiananagam et al.
Among several different inflammatory biomarkers, CRP is said to independently predict mortality in CKD patients
These findings are in agreement with those of Abraham et al.
Zahran et al.
Assessment of the kidney function tests revealed that serum creatinine level reached the peak of elevation in the CKD group followed by ESRD and both groups showed highly significant increase when compared to control group (6.28 ± 2.79, 5.81 ± 2.49 , 0.67 ± 0.19 respectively P=0.001). Also serum level of urea reached the peak of elevation in the CKD group followed by ESRD and both groups showed highly significant increase (P= 0.001) compared to control group(140.54 ± 71.48, 118.65 ± 23.90 m , 23.27 ± 6.69 respectively).
Comparison between CKD, ESRD and control groups as regards mean age values (51.72±12.70 ; 56.41±11.62 ; 28.90 ± 6.83 respectively ) and sex distribution revealed statistically significant difference between the three groups with more prevalent age and males distribution among ESRD group than other 2 groups (P=0.001). Comparison between CKD and ESRD groups as regard sex distribution revealed a highly statistical significant difference with more prevalent females in CKD group. Most of other studies on neutrophil apoptosis like revealed no statistical significant difference as regard demographic data
So regarding our study in uremic patients there was an increased BAK gene expression which is proapoptotic gene to a higher level in CKD group than ESRD group. On the other hand there was a decrease in expression level of bcl2 gene which is antiapoptotic gene with a level lower in ESRD compared to CKD group. These finding suggested a role of these two genes in acceleration in rate of neutrophil apoptosis and hence decrease in their life span. These finding through the light on the role of evaluated genes in survival and apoptosis of the studied groups and suggested their impact in controlling the inflammatory mechanisms and eventually their therapeutic potential.
All procedures performed in this study were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study is approved by Theodor Bilharz Research Institute ethical committee (TBRI-IRB number 01/16).
This study was processed in Kasr Al Aini main laboratory & TBRI, Hematology department. We thank our patients for their willing participation in our research.