INTRODUCTION:

Hypertension is one of the risk factor of chronic kidney disease and responsible for the stages of renal failure. Genetically, hypertension is a complex disorder with interaction of several genes and environmental factors 1. Most of the patients approximately, 25-40% of these subjects may develop Chronic Kidney Disease (CKD) and the end stage renal disease (ESRD) accompanied by hypertension2. Patients with hypertension and CKD is related to sympathetic nervous system uncontrolled activation.

The association between chronic kidney diseases and hypertension was well documented through various researches, but the relationship between these two has not been studied yet. Moreover, Renin Angiotensin System (RAS) is responsible for blood pressure regulation and kidney functions and their related activities. Angiotensin converting enzyme (ACE) is a zinc metallopeptidase found on the surface of endothelial and epithelial cells. When assessing the effect of RAS encoded genes, angiotensin converting enzyme (ACE) gene is found to be clinically prevailing and connecting to CKD3. Even though the factor relating ACE and CKD are not clear, certain types of RAS blockers are given to treat CKD patients to reduce severe kidney diseases. It is also unknown to find the response depending on individual pathophysiology.

The human ACE gene is located on chromosome 17q23, spans 21 kb, and includes 26 exons and 25 introns. In the National Center for Biotechnology Information (NCBI) records, more than 160 ACE gene polymorphisms are listed, most of which are single nucleotide polymorphisms (SNPs). Only 34 of those polymorphisms are located in coding regions; 18 of them are missense mutations. Its coding sequence is 4.3 kb in length and it codes for a 1306 amino acid in Angiotensin II Receptor 1 protein.

In order to determine ACE levels in plasma and tissues, ACE insertion/deletion polymorphism are the methods exist4. ACE gene contains 26 exons and 25 introns on chromosome 17q23. The gene has two alleles (D and I) based on Alu repetitive sequence in 16 intron and therefore shows three genotypes DD, DI and II5. Insertion (I) or deletion (D) in these 16th intron of ACE gene is responsible for hypertension related kidney diseases6, 7. Indeed, the D allele plays a more efficient role in getting damage the renoprotective action of ACE inhibitors7. The present case-control study was designed as an attempt to identify the possible associations between polymorphism of the ACE1 gene in patients with hypertension and CKD in the population of Tiruchirappalli.

MATERIALS AND METHODS:

Study subjects:

A total of 20 participants were selected and divided into four groups: group 1 patients with hypertension without chronic kidney disease, group 2 patients with chronic kidney disease without hypertension, group 3 patients with both chronic kidney disease and hypertension, group 4 patients with healthy individuals without any renal complications or hypertension.

Isolation and determination of DNA:

Blood samples (5.0 ml) were drawn from the peripheral vein of cases and controls into heparinized tubes. Genomic DNA extraction from the samples was performed by the standard salting out method. Agarose gel electrophoresis of blood genomic DNA was carried out8. DNA was then quantified by the method described by Sambrook et al. (1989)9.

Quantity of DNA (ng/µl) = OD at 260 nm X 50 X Dilution factor

1000

Designing of primers:

Primers specific for ACE 1 genes were sequenced and designed by Tabie et al. (2013)10 as shown in Table 1.

Table 1: Primers for Nested PCR amplification of ACE 1 gene.

Oligo Name	Length	Tm(°C)	Gc%	Sequence
FP1	22	56.7	54.6	5’-CTGGAGACCACCCATCCTTTCT-3’
RP1	25	57.7	48	5’-GATGTGGCCATCACATTCGTCAGAT-3’
FP2	24	65.9	70.8	5’-TCGGACCACAGCGCCCGCCACTAC-3’
RP2	23	62.4	65.2	5’-CGCCAGCCCTCCCATGCCCATAA-3’

*FP1, RP1 – Forward and reverse primers for first PCR amplification; FP2, RP2 – Forward and reverse primers for second PCR amplification.

PCR amplification of ACE1:

About 5 µl aliquot of PCR amplified product was loaded on 2% agarose in 1X TAE buffer and at 50V for 45 minutes. 100 bp DNA ladder was used as the marker and the PCR products were visualized in a UV transilluminator. PCR amplification of deletions (D) and insertions (I) of ACE1 were evaluated in a 20μl reaction mixture containing 200ng of the template DNA, 7.5 pmol/l of each primer, 0.2mM of each dNTP, 1.5mM MgCl2, 2.5 µL10X buffer and 1U Taq DNA polymerase. The PCR amplification included 30 cycles of denaturation at 94°C (one minute), annealing at 58°C (one minute) and extension at 72°C (two minutes) as shown in Table 2.

Table 2: PCR Cycling Conditions.

Step	Temperature	Time
Initial denaturation	94° C	1 minute
Denaturation (for cycles)	94° C	30 seconds
Annealing (for cycles)	58° C	30 seconds
Extension (for cycles)	72° C	1 minute
Final extension	72° C	8 minutes
Number of cycles -30

Statistical analysis:

All statistical analysis was performed with SPSS 14.0 for Microsoft Windows. Group findings were compared with chi squared test (P ≤ 0.05). The association between ACE gene polymorphism with hypertension and CKD, Odds ratio were analyzed with (P≥ 0.05).

RESULTS AND DISCUSSION:

DNA isolated from blood samples of the study group was confirmed by agarose gel electrophoresis which was loaded in lane 3, 4, 5 and 6 and the size was found to be 23 kb as compared with the Lamba DNA/Hind III digest used as marker loaded in lane 1 as shown in Figure 1.

Figure 1: Agarose gel electrophoresis of human blood sample shown as a representative.

With the electrophoretic results, DNA purity and quantity were assessed by absorbance. The quantity of DNA for patients who belonged to group 1 was 5ng/μl, group 2 was 7.5ng/μl, group 3 was 15ng/ μl and group 4 was 12.5ng/μl as shown in Table 3.

Table 3: Quantification of DNA for four groups

Group	OD at 260nm	Quantity of DNA (ng/μl)
1	0.01	5
2	0.02	7.5
3	0.03	15
4	0.05	12.5

Nested PCR with first reaction product were done as shown in Figure 2 and 3 where PCR lane 1 was loaded with 100bp DNA ladder (marker) and lane 3 to 6 were loaded with samples from group 1 to group 4. Band in DNA ladder was obtained with I allele at 490 bp and D allele at 190 bp region. A band of 490 bp represented II genotype, 190 bp represented DD genotype and two bands of 490 bp and 190 bp demonstrated ID genotype (Figure 2). PCR result with sample groups showed bands with homozygous DD with II genotypes was absent, whereas two bands of 190 bp and 335 bp were obtained for ID genotype (Figure 3).

Figure 2: Nested PCR with first reaction product by Agarose gel electrophoresis.

Figure 3: Nested PCR with sample second reaction product by Agarose gel electrophoresis.

In the present study, there was ACE1 polymorphism which was checked in study groups using a nested PCR reaction. In the first PCR, a band for I allele at 490 bp and for D allele at 190 bp was obtained. In lane 3 and 5 there were 2 bands one at 190 bp and another at 490bp which represented ID genotype, in lane 4 there was only one band at 190 bp which represented DD genotype and in lane 6 also there was only one band at 490 bp which represented II genotype. In the second PCR, no bands resulted for homozygous DD and II genotypes whereas two bands of 190 bp and 335 bp illustrated ID genotype. When compared genotype distribution of ACE1 gene, no significant differences were found between four groups as shown in Figure 4.

Figure 4: Genotype and allele distribution of ACE1 gene polymorphisms in four groups.

According to the study done by Tabei et al. (2013), their findings compared the pertinence of AGT-TT genotype in patients with hypertension and simple renal cysts10. The results confirmed the additive role of AGT gene in renin-angiotensin system during the mechanism of hypertension and simple renal cysts formation. There were prevalent in different genetic polymorphisms of ACE1 among different groups but none of the genotype distributions followed the Hardy-Weinberg equilibrium. Similarly in the present study, the statistical analysis revealed that none of the genotype distributions followed the Hardy-Weinberg equilibrium. The overall frequency of II genotype, DD and ID were0.226, 0.499 and 0.276 respectively. Similarly, the frequency of I allele in the study population was 0.475 and D allele was 0.525 respectively. The differences in genotypes or alleles were not statistically significant.

The results of previous research were in accordance with the results of the present findings. The conventional PCR method in genotyping ACE gene polymorphism was variable, required careful control, and often needed repeated testing, especially to verify the ID heterozygote. The confirmatory PCR method had higher accuracy but needed preferential amplification of the multiplexed PCR. The confirmatory PCR could be the method of choice in screening ACE gene polymorphisms11. In the present findings the use of conventional and confirmatory PCR to rule out the genetic polymorphism was in agreement with the previous study.

In order to quantify strongly the association between ACE gene polymorphism with hypertension and CKD, Odds ratio were analyzed with (P≥ 0.05). Results of Odds Ratio (OR) analysis showed the ID genotype in significant relation with hypertension and CKD. As a result of OR analysis, OR of II genotype was equal to 1, which confirmed that the polymorphism has no influence on hypertension or CKD. The OR of ID genotype found to be greater than 1 and therefore, the ID genotype is associated with the development and progression of hypertension and CKD. OR of DD genotype was found lesser than 1 and consequently, the gene is less significant in determining the occurrence or progression of the CKD or hypertension as shown in Table 4.

Table 4: Odd Ratio (OR) analysis of ACE1 gene polymorphism.

Independent Variant	Genotype	OR			P value
Independent Variant	Genotype	Cases	Control	Result	P value
ACE-1	II	3	1	1	0.517
	ID	9	2	1.5	0.435
	DD	3	2	0.5	0.371

Statistical method with (P ≥ 0.05) showed that there were no significant difference between groups and genotype versus genotype distribution related to ACE gene. Although the results comprise the evidence of an additive role for the ID genotype of the ACE1 gene in the process of hypertension and chronic kidney disease formation in the study population, large case-control studies should be performed to verify the research findings. In order to quantify strongly the association between ACE gene polymorphism with hypertension and CKD, Odds ratio were analyzed. Results of Odds Ratio (OR) analysis showed the ID genotype in significant relation with hypertension and CKD. As a result of OR analysis, OR of II genotype was equal to 1, which confirmed that the polymorphism has no influence on hypertension or CKD. The OR of ID genotype found to be greater than 1 and therefore, the ID genotype is associated with the development and progression of hypertension and CKD. OR of DD genotype was found lesser than 1 and consequently, the gene is less significant in determining the occurrence or progression of the CKD or hypertension. Even statistical method with (P ≥ 0.05) showed that there were no significant difference between groups and genotype versus genotype distribution related to ACE gene.

CONCLUSION:

ACE1 variant patients with chronic kidney disease and hypertension have a significant role in the pathophysiology. The results showed the differences in other genotypes or alleles were not statistically significant with exception of ID genotype. Although the results comprise the evidence of an additive role for the ID genotype of the ACE1 gene in the process of hypertension and chronic kidney disease formation in the study population, large case-control studies should be performed to verify the research findings.

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Received on 11.05.2017 Modified on 05.06.2017

Research J. Pharm. and Tech. 2017; 10(8): 2607-2610.

DOI: 10.5958/0974-360X.2017.00462.0