Study of Oxidative Stress Marker 8-hydroxy-2’-deoxyguanosine in Chronic Kidney Disease Patients

Introduction

Renin-Angiotensin system (RAS) has been implicated in pathological changes of organ damage through modulation of gene expression, proliferation, and inflammatory response. ACE is a key component of renin-angiotensin system (RAS). ACE I/D polymorphism varies as per individual, ethnicity, geography and is associated with common diseases like hypertension, coronary heart disease, and nephropathy. Uraemic state in case of CKD patients causes impairment of DNA damage repair resulting in chromosomal damage. Such damage can also be due to the formation of reactive oxygen species because of chronic inflammation. Reactive Oxygen and nitrogen species in CKD patients can lead to DNA strand breaks, point mutations, and aberrant DNA cross-linking causing genomic instability.^1-2

Various types of inflammatory markers such as interleukin 6 and tumor necrosis factor alpha have been studied in CKD patients. Oxidative stress markers like 8-hydroxy-2’-deoxyguanosine (8-OHdG), advanced glycation end products, advanced oxidation protein products, and Malondialdehyde have been also studied in detail. Such clinical biomarkers help to stage the CKD patients. 8-hydroxy-2’-deoxyguanosine is a promutagenic oxidation product of guanine. Its level reflects nuclear damage due to small oxidative changes and can give rise to G to T transversion mutations in key genes known to be involved in the development of cancer.^3-4

Materials and Methods

A 5 mL venous blood sample was taken from all patients before starting hemodialysis with all aseptic precautions in EDTA vacutainers.

Patients available in the medicine department, diagnosed with CKD for a period of 12-15 months were included in the study. Patients with co-existing other illnesses, cancer patients, patients on chemotherapy, or drugs likely to cause DNA damage were excluded.

The patients who gave informed consent were included in the study. The ethical clearance was taken from the BIMS Institutional Ethical Committee.

DNA Extraction and PCR

DNA was extracted from whole blood containing ethylenediaminetetraacetic acid (EDTA) by Qiagen kit method. The quality and quantity of the DNA was analyzed by biosphectrometer. To determine the ACE genotype, the genomic DNA fragment of the intron 16 of ACE gene was amplified by PCR. The conditions for amplification were Initial denaturation: 94ºC for 5 min, Denaturation: 94ºC for 30 s, Annealing: 58ºC for 45 s, Extension: 72ºC for 45 s, Cycling condition: 30 cycles, Final extension: 72ºC for 7 min and Hold at 4ºC. Primers for ACE Polymorphism were used in this study. The flanking primer sequences as reported by Rigat et al ⁵ were;

Forward Primer: CTG GAG ACC ACT CCC ATC CTT TCT (50 nmol)

Reverse Primer: GAT GTG GCC ATC ACA TTC GTC AGAT (50 nmol)

Once the amplicons were obtained, they were subjected to 2% agarose horizontal gel electrophoresis with ethidium bromide, and the bands were visualized under UV light. With the help of DNA ladder, Deletion (D allele) and Insertion (I allele), were identified at 191 and 478 bp fragments, respectively.

Oxidative stress assay

Serum 8-OHdG (8-Hydroxyguanosine) was measured using a commercially available competitive ELISA kit (Genetix Biotech Asia Pvt. Ltd, INDIA) by diluting the samples. The kit can measure 8-OHdG values ranging from 0.94 to 100 ng/mL, using a monoclonal-specific antibody. The protocol mentioned in the kit was followed.

Statistical analysis

The data obtained were represented in mean ± SD. The parametric data was analysed by unpaired ‘t’ test and non-parametric data was analysed by Mann Whitney ‘U’ test.

Results

Graph 1 shows the standardization calibration graph of the 8-OHdG enzyme assay kit. 

Table no 1 shows the mean±SD values of ACE gene alleles with respect to the number of haemodialysis and serum 8-OHdg levels studied.

Table no 2 shows the statistical comparison (p=value) of II ACE gene alleles with other alleles in respect of the numbers of haemodialysis and serum 8-OHdg levels studied.

The total number of cases studied was 47. On PCR amplification and gel electrophoresis, 17 cases showed the presence of ACE gene DD allele, 12 showed II allele,18 showed DI allele in ACE gene as shown in Table 1. In the case of the above subgroups, the mean ± SD was calculated for the number of haemodialysis, and concentration of level of 8-OHdG (ng/ml) by ELISA kit as shown in the table. Initially, the standardization calibration graph of 8-OHdG enzyme assay kit was done as shown in Graph 1. In the case of DD allele, the mean ± SD for the number of haemodialysis done was found to be 185.82±131.69 and the mean concentration level of 8-OHdG (ng/ml) by Elisa kit was found to be 39.02±29.5 ng/mL.

In the case of II allele, the mean ± SD for the number of haemodialysis done was found to be 339.33±261.59 and the mean concentration level of 8-OHdG (ng/ml) by ELISA kit was found to be 15.74±11.76 ng/mL. In the case of DI allele, the mean ± SD for the number of haemodialysis done was found to be 183.33±182.93 and the mean concentration level of 8-OHdG (ng/ml) by Elisa kit was found to be 25.39±23.06 ng/mL as shown in Table 1.

Table 2 shows the comparison of mean values for statistical significance between ACE gene II alleles in comparison to other alleles regarding the number of haemodialysis and serum 8-OHdg levels studied. From the table, it is clear that there was no statistically significant difference seen in the comparison between ACE gene II allele values with DD and D/I gene alleles with respect to the number of haemodialysis and 8-OHdG levels.

Discussion

ACE gene play’s role in hypertension and secondarily affects kidney. This has been studied by many researchers. The ACE I/D polymorphism may invite the utmost risk for growing CKD in hypertensive patients, especially Asian males. Chin Lin et al., studied the ACE genotype and its alleles (DDII and D/I) and found that CKD threat was elevated with the D allele as compared to I allele in Asian society and hypertension had affirmative moderate effects.⁶ The males were at higher risk where the D allele showed 3.75 fold greater risk for CKD than I allele in hypertensive cases. A similar study done by Taposh Sarkar et al., found that the development of CKD is linked to DD genotype of ACE gene within hypertensive patients.⁷

Apart from genetic factors, morbidity in patients with CKD can be due to non-genetic factors such as suppression of the immune system, viral associated factors, increased levels of oxidative stress, chronic inflammation, accumulation of uremic toxins and reduced antioxidant levels.^8-9

In our study, we did not find any statistical difference between the oxidative stress marker levels in different ACE gene allele patients. Molnar GA et al., observation concluded that in carriers of allele D, the serum level of angiotensin-II was higher, which can be associated with increased oxidative stress and subsequent endothelial damage.¹⁰ Allele D patients have also been found with higher activity of gamma-GT and higher albuminuria. Based on this, D allele may contribute via increased glycation and oxidative stress to the target organ damage in type 2 diabetes. A study done by Deepashree G A et al., showed that the DD genotype of ACE gene had a significant association with the advancement of CKD in Diabetic nephropathy (OR=0.37; 95 % CI=0.14–0.94; p=0.033).¹¹

Even the type of haemodialysis affects the level of oxidative stress markers. In case of haemodialysis patients oxidized LDL, homocysteine and plasma thiobarbituric acid reactive substances were found at a higher level than peritoneal hemodialysis patients however some studies have also reported contradictory findings. The associated diseases, mainly like diabetes, can influence the level of oxidative stress markers. 8-OHdg significantly correlated with other oxidative stress markers. The study of 8-OHdg is significant because it is a marker for intracellular oxidative stress.^12,13-17

8-OHdg can be measured by different methods like GCMS, HPLC, FPG-comet assay, and ELISA. Results from these methods differ, GC-MS measures the highest while the FPG- comet assay the lowest. To improve 8-OHdg quantifications, the European Standard committee on Oxidative DNA damage was established. On the same lines, European standard committee on urinary lesion analysis was established for the study of Urinary 8-OHdg levels.^18-20

Limitation of study

Large sample survey is needed for sequencing of ACE gene which is not done in the present study.

Conclusion

In this study, it was found that the hemodialysis requirement did not differ in CKD patients having different ACE gene alleles. The oxidative stress marker 8-OHdG also did not show significant changes in different ACE gene allele groups.

Conflict of Interest

Nil

Acknowledgement

Authors are thankful to Director, Advance Research Wing, RGUHS, Bengaluru, Karnataka, for providing funding, which was crucial to the success of this project, and thankful to Dr Kishore Bhat for unwavering support, encouragement, and access to facilities to complete the project.