Chronic kidney disease is increasingly understood as a systemic disorder involving not only progressive loss of filtration capacity but also persistent metabolic, inflammatory and molecular alterations. Oxidative stress is a central feature of CKD and becomes progressively more pronounced with deterioration of renal function.¹

Despite severe oxidative stress, the antioxidant response in advanced renal disease may be paradoxically impaired. A major regulator of this response is nuclear factor erythroid 2-related factor 2, encoded by NFE2L2. Under oxidative conditions, Nrf2 activates antioxidant response element-dependent genes, including HMOX1, NQO1 and genes responsible for endogenous antioxidant defence.²˒³

Experimental evidence indicates that activity of the Nrf2–Keap1 pathway is impaired in chronic renal failure. Kim and Vaziri demonstrated reduced Nrf2 activity together with increased Keap1 and diminished downstream antioxidant enzymes, suggesting failure of an important cytoprotective pathway.²

Epigenetic mechanisms provide a biologically plausible explanation for persistent dysregulation of antioxidant gene expression. DNA methylation can alter transcription without changing the nucleotide sequence and is influenced by inflammation, uraemia, homocysteine metabolism and oxidative stress.⁴˒⁵

The present study evaluated global DNA methylation and Nrf2-related antioxidant gene expression in CKD and investigated their association with oxidative stress and disease severity.

The study used a hospital-based analytical case–control design and included 60 patients with CKD and 60 age- and sex-matched healthy controls.

Blood samples were collected after overnight fasting. Clinical assessment included renal diagnosis, duration of CKD, dialysis status, comorbidities and treatment history. Renal function was assessed using serum creatinine, blood urea and CKD-EPI-derived eGFR.

Genomic DNA was obtained from peripheral blood leukocytes. Global DNA methylation was quantified as the percentage of 5-methylcytosine using a validated ELISA-based methylation assay.

Messenger RNA expression of genes involved in the Nrf2-mediated antioxidant pathway was assessed using molecular methods described in the thesis protocol. Genes of interest included NFE2L2/Nrf2, HMOX1/HO-1, NQO1, SOD1, CAT and GPX1.

MDA and 8-OHdG were assessed as indicators of lipid peroxidation and oxidative DNA damage respectively. CKD patients were analysed according to disease stage and maintenance haemodialysis status. Relationships among methylation, gene expression, renal function and oxidative stress were determined using appropriate correlation analysis. Statistical significance was set at p<0.05.

Global DNA methylation was significantly reduced among CKD patients.

 Table 1. Global DNA methylation in CKD and controls

 Table 2. Global methylation according to dialysis status

The lowest global methylation was observed among maintenance haemodialysis patients.

 Table 3. Expression of Nrf2/antioxidant pathway genes

NFE2L2 demonstrated the largest relative reduction.

Table 4. Global DNA methylation across CKD stages

Correlation analysis demonstrated that eGFR correlated positively with global 5-mC (r=+0.57, p<0.001). MDA correlated negatively with global 5-mC (r=−0.55, p<0.001), and 8-OHdG showed a similar inverse relationship (r=−0.51, p<0.001). MDA was inversely associated with NFE2L2 expression (r=−0.46, p<0.001).

The principal finding of this study was a significant reduction in global DNA methylation among CKD patients accompanied by suppressed expression of several components of the Nrf2-mediated antioxidant pathway.

Global 5-mC was significantly lower in CKD patients than healthy controls and demonstrated a progressive reduction across CKD stages. The greatest alteration occurred among Stage 5D haemodialysis patients. This pattern suggests that the uraemic environment, inflammation and advanced renal dysfunction may influence the epigenetic landscape.

Hsueh et al. demonstrated an association between global DNA methylation and CKD, supporting the concept that methylation abnormalities participate in renal disease.⁶ Genome-wide investigations have similarly demonstrated multiple differentially methylated loci in patients with kidney failure.

Published results are not completely uniform. Hsu et al. reported no significant increase in global methylation among chronic haemodialysis patients, although changes in DNA methyltransferase expression were detected.⁷ Conversely, Ghigolea et al. demonstrated differences in global DNA methylation among patients receiving haemodialysis and haemodiafiltration.⁸ These divergent findings indicate that epigenetic patterns may be affected by dialysis modality, inflammatory state, nutritional factors and laboratory methodology.

A second major observation was reduced expression of NFE2L2 and downstream antioxidant genes. Nrf2 functions as a master transcriptional regulator of cellular defence against oxidative stress. Kim and Vaziri demonstrated impairment of the Nrf2–Keap1 pathway in chronic renal failure.² Subsequent studies have reinforced the importance of impaired Nrf2 signalling in renal inflammation and oxidative stress.³˒⁹

Juul-Nielsen et al., in a systematic review of the NRF2 system in human CKD, found reduced NRF2 in the majority of eligible clinical studies, although results for individual downstream targets were more heterogeneous.¹⁰ The reduced NFE2L2 expression in the present study therefore agrees with the predominant pattern reported in human CKD.

The inverse associations between MDA and global methylation and between MDA and NFE2L2 expression are particularly noteworthy. They suggest that increasing oxidative stress occurs alongside progressive epigenetic and transcriptional dysregulation. However, because the study is observational, the direction of causality cannot be determined.

The study is also limited by the use of peripheral blood rather than renal tissue and by its single-centre case–control design. Future longitudinal studies incorporating locus-specific methylation, protein expression and clinical outcomes would help determine the prognostic relevance of these observations.

Patients with CKD demonstrate significant global DNA methylation abnormalities and reduced expression of NFE2L2 and multiple downstream antioxidant genes. These abnormalities become increasingly pronounced with worsening renal function and maintenance haemodialysis. Their close relationships with MDA, 8-OHdG and eGFR support an interaction between oxidative injury and epigenetic dysregulation in CKD. Global 5-mC and Nrf2-pathway gene expression warrant further evaluation as potential molecular biomarkers of CKD severity.

1. Daenen K, Andries A, Mekahli D, Van Schepdael A, Jouret F, Bammens B. Oxidative stress in chronic kidney disease. Pediatr Nephrol. 2019;34(6):975-991. doi:10.1007/s00467-018-4005-4.
2. Kim HJ, Vaziri ND. Contribution of impaired Nrf2-Keap1 pathway to oxidative stress and inflammation in chronic renal failure. Am J Physiol Renal Physiol. 2010;298(3):F662-F671. doi:10.1152/ajprenal.00421.2009.
3. Ruiz S, Pergola PE, Zager RA, Vaziri ND. Targeting the transcription factor Nrf2 to ameliorate oxidative stress and inflammation in chronic kidney disease. Kidney Int. 2013;83(6):1029-1041. doi:10.1038/ki.2012.439.
4. Jin J, Chen H, Meng XM. Editorial: epigenetic regulation in renal development, physiology and disease. Front Physiol. 2022;12:818190. doi:10.3389/fphys.2021.818190.
5. Perna AF, Ingrosso D. Homocysteine and chronic kidney disease: an ongoing narrative. J Nephrol. 2019;32(5):673-675. doi:10.1007/s40620-019-00622-1.
6. Hsueh YM, Chen WJ, Lee HL, et al. Global DNA methylation and the association between metal exposure and chronic kidney disease. Front Public Health. 2023;11:1104692. doi:10.3389/fpubh.2023.1104692.
7. Hsu CY, Sun CY, Lee CC, Wu IW, Hsu HJ, Wu MS. Global DNA methylation not increased in chronic hemodialysis patients: a case-control study. Ren Fail. 2012;34(10):1195-1199. doi:10.3109/0886022X.2012.723280.
8. Ghigolea AB, Moldovan RA, Gherman-Caprioara M. DNA methylation: hemodialysis versus hemodiafiltration. Ther Apher Dial. 2015;19(2):119-124. doi:10.1111/1744-9987.12238.