Ethical recognition of research
Before being included in the study, all individuals gave written informed consent, and blood samples were taken according to protocols approved by the hospital’s Outpatient Clinic’s Ethics Committee.
Study populations
Patients with CKD were screened and diagnosed between September 2018 and March 2019 from the Outpatient Clinic of the hospital. The study involved a total of 216 Egyptian adults, including 116 patients suffering kidney pathologies at different stages, and 100 controls. The participants were divided as follow:
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(1)
ESRD group: this group included 100 females and males ESRD patients whose ages ranged from 18 to 82 years old (mean age = 47 years and SD = 16.1).
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(2)
CKD group: this group included 16 females and males CKD patients whose ages ranged from 15 to 58 years old (mean age =40 years and SD = 11.09). The clinical data of all patients were obtained from medical records. Patient's data included age, sex, and laboratory investigation such as Hb conc., creat conc., bilirubin, albumin, SGPT, SGOT, FBS, HBS Ag, and HCV.
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(3)
Control group: this group included 100 healthy samples (54 females and 46 males) for comparison; their age and sex were matched with the chronic kidney disease patients, and their families did not have any record of chronic kidney disease history (mean age = 37 years, SD = 12.7).
Sampling
Three milliliters of peripheral blood was collected from all subjects using vacationers and transferred to test tubes containing EDTA solution as an anticoagulant to obtain whole blood, for extraction and purification of DNA. Peripheral blood samples are also utilized to investigate biochemical markers related to CKD.
DNA extraction and purification
The handling and extraction of genomic DNA for all participants enrolled in this work was accomplished by using the generation DNA purification capture column kit (BioFlux, China). The extraction procedures were carried out based on the prescripts provided by the producer for the peripheral blood samples.
DNA in the sample was released using proteinase K (PK) solution and lysis B buffer. The released DNA was bound exclusively and specifically to the biospin membrane in the presence of lysis B buffer and ethanol under the appropriate salt iron and pH conditions. Denatured protein and other contaminants were removed with twice washing procedures. With the elution buffer, the DNA was then eluted from the membrane [17].
The amplification and genotyping of eNOS (rs 2070744) and MnSOD Val16Ala (rs4880) Gene variants were described using the tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) [18].
Genetic analysis
Determination of MnSOD genotype
T-ARMS-PCR was performed by using the tetra primer (forward inner primer, reverse inner primer, forward outer primer, reverse outer primer) and the amplicons purification using QIAquick PCR purification kit (Qiagen) [19].
The DNA was amplified using specific oligonucleotide primers based on the published sequence (Biolegio, Nijmegen-The Netherlands). Two primer pairs were used to accentuate and designate the genotype of a deoxyribonucleic acid fragment, as well as the Ala16Val SNP within the human MnSOD sequence. The 3′-end of the allele-specific primers were underlined
F1 (forward) 5′-CACCAGCACTAGCAGCATGT-3′;
F2 (forward)5′GCAGGCAGCTGGCTaCGGT-3′;
R1 (reverse) 5′-ACGCCTCCTGGTACTTCTCC-3′
and R2 (reverse)5′-CCTGGAGCCCAGATACCCtAAAG-3.
PCR was conducted in an exceedingly total volume of 25 μL containing 4 μL of genomic DNA because the templet, 4 μL of every primer, 13 μL Master Mix (2×). PCR amplification was conducted with primary denaturation at 95 °C for 2 min, followed by 35 cycles of 1 min of denaturation at 94 °C, 1 min of hardening at 60 °C, 1 min of extension at 72 °C, and a further a pair of min extension at 72 °C at the tip of the ultimate cycle. Next, The PCR products were electrophoresed on 2% agarose gel and visualized using ethidium bromide under ultraviolet illumination below ultraviolet radiation.
The PCR product for MnSOD cistron was detected at 189 bp for wild-type (Val/Val), and 366 bp for mutant type (Ala/Ala) then photographed by the photographic camera. Once the sample had a pair of bands at 189 bp in the wild tube and 366 bp within the mutant tube, it meant it had Val/Ala heterozygote genotyping. On the opposite hand, once the sample had a band that appeared at 189 bp in the wild-type tube and no band within the mutant factor, this meant it had Val/Val homozygote genotyping. Once the sample had a band at 366 bp within the mutant factor and no band in the wild-type factor, this meant it had Ala/Ala homozygote genotyping as shown in Fig. 2.
Determination of eNOS genotype
PCR was performed by using only one pair of primers, 2 oligonucleotide primers that flank the region of the 27-bp repeat sequence in intron 4 of the eNOS gene were used for polymerase chain reaction (PCR) amplification. The eNOS primers were synthesized by Integrated DNA Technologies, Inc. (IDT, Santa Clara, CA, USA). The forward primer was 5′-AGGCCCTATGGTAGTGCCTTT-3′ and the reverse primer was 5′-TCTCTTAGTGCTGTGGTCAC-3′ [20].
PCR was conducted in an exceedingly total volume of 25 μL in only one tube containing 4 μL of genomic DNA, 4 μL of every primer, 13 μL Master Mix (2×). The reaction mixture was heated to 95°C for 6 min for denaturation and then subjected to 35 cycles at 95 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 2 min, followed by a final extension step at 72 °C for 7 min.
The PCR products were electrophoresed on 2% agarose gel and visualized using ethidium bromide under ultraviolet illumination. The PCR product for the eNOS gene (intron 4a/4b) was detected at 420 bp for wild-type (4b) and 393 bp for mutant-type (4a) then photographed by the digital camera.
When the sample had 2 bands at 420 bp wild-type 4b allele and 393 bp for mutant-type it meant it had 4ab heterozygote genotyping. On the other hand, when the sample had a band at 393 bp for mutant-type and no band at 420 bp for wild-type, this meant it had 4aa homozygote genotyping. When the sample had a band at 420 bp for wild-type and no band for mutant-type, this meant it had 4bb homozygote genotyping as shown in Fig. 3.
Statistical analysis
Data were collected, coded, revised, and entered into the Statistical Package for science (IBM SPSS) version 20. For qualitative data, mean, standard deviations, and ranges were used; for quantitative data with a parametric distribution, median with interquartile range (IQR) was used; and for quantitative data with a non-parametric distribution, median with interquartile range (IQR) was used.
When comparing two groups with qualitative data, the chi-square test was utilized, and the Fisher exact test was used instead of the chi-square test when the expected count in any cell was less than 5. The independent t test was used to compare two groups with quantitative data and a parametric distribution, while the Mann-Whitney test was used to compare 2 groups with qualitative data and a non-parametric distribution.
The one way analysis of variance (ANOVA) test was used to compare more than two groups with quantitative data and parametric distribution, and the Kruskal-Wallis test was used to compare more than 2 groups with quantitative data and non-parametric distribution.
The margin of error acceptable was set to five because the confidence interval was set at 95%. Because of the following, the P value was deemed significant: P > 0.05: non-significant (NS), P < 0.05: significant (S), P < 0.01: highly significant (HS). Figure 4 illustrates in silico data analysis.
