Chronic Kidney disease (CKD) is renal damage for ≥3 months, defined by structural or functional deformities of renal (clinical deformities or deformities of imaging or the structure of blood), with or without reduced GFR. ^[1] CKD is also described as GFR <60 ml/min/1.73m for ≥3 months, with or without kidney damage. There are currently five stages of CKD. ^[2] Chronic kidney diseases are a global public health problem associated with premature mortality, decreased quality of life. A trend towards an increase in its incidence and prevalence has been reported worldwide. ^[3]

The stages of CKD are typically classified based on the estimated glomerular filtration rate (eGFR), which measures how efficiently the kidneys filter waste from the blood. ^[4] The stages range from stage 1 (mild kidney damage with normal or slightly reduced eGFR) to stage 5 (end-stage renal disease, or ESRD, with severely reduced kidney function requiring dialysis or kidney transplantation for survival). ^[5]

Common causes of CKD include diabetes, hypertension (high blood pressure), glomerulonephritis (inflammation of the kidney's filtering units), and polycystic kidney disease (a genetic disorder causing fluid-filled cysts to form in the kidneys). ^[6]

CKD is associated with an increased risk of complications such as cardiovascular disease, anemia, bone disorders, and fluid overload. ^[7] Early detection and management of CKD, including lifestyle modifications (e.g., diet, exercise), medication management (e.g., blood pressure control, treatment of underlying conditions), and regular monitoring, can help slow disease progression and reduce the risk of complications. ^[8]

Chronic kidney disease (CKD) affects millions of people worldwide, with an increasing prevalence due to aging populations and rising rates of conditions such as diabetes and hypertension. ^[9] CKD is characterized by a gradual decline in kidney function over time, leading to complications such as cardiovascular disease, anemia, and bone disorders. ^[10] One of the hallmarks of CKD is the presence of microalbuminuria, defined as the excretion of small amounts of albumin in the urine. ^[11]

Oxidative stress results from an imbalance between the production of ROS and the body's antioxidant defenses. ROS, including superoxide radicals, hydrogen peroxide, and hydroxyl radicals, are generated during normal cellular metabolism and play essential roles in signaling pathways and host defense mechanisms. However, excessive ROS production can overwhelm antioxidant systems, leading to oxidative damage to lipids, proteins, and DNA. ^[12]

Antioxidants play a crucial role in protecting cells from oxidative stress, which occurs when there is an imbalance between reactive oxygen species (ROS) production and antioxidant defenses. In CKD patients, oxidative stress is heightened due to various factors, including inflammation, uremic toxins, and impaired antioxidant systems. Understanding the correlation between antioxidant status and microalbuminuria in CKD patients is essential for elucidating the mechanisms underlying disease progression and identifying potential targets for intervention. ^[13]

Aim and Objectives

Correlation Between Antioxidant Status and Microalbuminuria in Chronic Kidney Disease Patients.

Study Design:     Observational or cross-sectional study.

Study Population: CKD patients from outpatient clinics or hospitals, Index Medical College. Patients diagnosed with CKD stages 1–5, based on the Kidney Disease Improving Global Outcomes (KDIGO) guidelines.

Duration: Study was done from January 2023 to December 2024.

Inclusion Criteria:

• Patients diagnosed with CKD stages 1-5.
• Age > 18 years.
• Patients willing to participate in the study.
•  

Exclusion Criteria:

• Patients with acute kidney injury.
• Patients with other major comorbidities (e.g., cancer, severe liver disease).
• Pregnant or lactating women.
• Patients on antioxidant supplementation (e.g., vitamin C, vitamin E) within the last 3 months.
• Patients with a history of kidney transplantation or on dialysis.

Ethical Considerations: Obtain approval from the Institutional Review Board (IRB) or Ethics Committee.

Informed Consent: Written informed consent will be obtained from all participants before enrollment. The consent form will include details about the study objectives, procedures, risks, benefits, and confidentiality.

Confidentiality: All patient data will be anonymized and stored securely. Identifiable information will be kept separate from research data.

Data Collection:

Demographic and Clinical Data: Collect information on age, gender, duration of CKD, comorbidities, medications, and lifestyle factors.

Antioxidant Status Assessment: Measure antioxidant biomarkers such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and total antioxidant capacity (TAC) using validated assays.

Other Laboratory Parameters: Measure serum creatinine, estimated glomerular filtration rate (eGFR), serum albumin, and other relevant biochemical markers.

Antioxidant Status Assessment

• Biomarkers Measured:
  1. Superoxide Dismutase (SOD):Measured using a colorimetric assay based on the inhibition of nitroblue tetrazolium (NBT) reduction.
  2. Catalase:Measured using spectrophotometry by monitoring the decomposition of hydrogen peroxide at 240 nm.
  3. Glutathione Peroxidase (GPx):Measured using an enzymatic assay that monitors the oxidation of NADPH at 340 nm.
  4. Total Antioxidant Capacity (TAC):Measured using the ferric reducing antioxidant power (FRAP) assay, which measures the reduction of ferric-tripyridyltriazine (Fe³⁺-TPTZ) to ferrous-tripyridyltriazine (Fe²⁺-TPTZ).
• Sample Collection:Blood samples will be collected in heparinized tubes, centrifuged at 3000 rpm for 10 minutes, and stored at -80°C until analysis.

Statistical Analysis:

Descriptive Analysis: Summarize demographic and clinical characteristics of the study population. Correlation Analysis: Assess the correlation between antioxidant status (SOD, catalase, GPx, TAC) using Pearson or Spearman correlation coefficients. Multivariate Analysis: Perform multivariable regression analysis adjusting for potential confounders (e.g., age, gender, eGFR) to determine independent associations. Subgroup Analysis: Explore correlations stratified by CKD stage or other relevant factors. Statistical Software: Utilize SPSS V29 statistical software for data analysis

Table 1: Demographic Characteristics of the Study Population

In table 1, the mean age of the study population is 55.3 years, with a standard deviation of 12.4 years, indicating a relatively wide age range. The study population is nearly evenly distributed between males (52%) and females (48%). The majority of participants (45%) are in CKD Stage 3, which is characterized by moderate kidney damage (eGFR 30–59 mL/min/1.73 m²). The mean duration of CKD is 6.2 years, with a standard deviation of 4.1 years, indicating variability in disease duration among participants. A significant proportion of participants (78%) have hypertension, which is a common comorbidity in CKD patients. Diabetes mellitus is present in 62% of participants, reflecting its role as a leading cause of CKD. Smoking is reported in 22% of participants, which is a modifiable risk factor for CKD progression and cardiovascular disease.

Table 2: Antioxidant Biomarker Levels in CKD Patients

In table 2, Mean Level of Superoxide Dismutase (SOD) is 12.3 ± 3.2 U/mL. Mean Level of Catalase is 45.6 ± 10.4 U/mL. Glutathione Peroxidase (GPx) Mean Level is 8.7 ± 2.1 U/mL and Total Antioxidant Capacity (TAC) were 1.2 ± 0.3 mmol/L.

Table 3: Correlation Between Antioxidant Biomarkers and Microalbuminuria

1. Negative Correlations:

• All biomarkers show a negative correlation, meaning as the variable increases, the biomarker levels decrease.
• The strongest correlation is seen in Total Antioxidant Capacity (TAC) (-0.40, p < 0.001), suggesting a significant inverse relationship.
• Glutathione Peroxidase (GPx) (-0.35, p = 0.001) also shows a strong inverse correlation.
• Superoxide Dismutase (SOD) (-0.32, p = 0.002) and Catalase (-0.28, p = 0.008) also have negative but slightly weaker correlations.

2. Statistical Significance (p-values):

• Since all p-values are < 0.05, the correlations are statistically significant, indicating a meaningful relationship between the biomarkers and the variable.

Table 4: Comparison of Antioxidant Levels Across CKD Stages

In table 5, Superoxide Dismutase (SOD) Levels: SOD levels decrease from 14.2 U/mL in Stage 1 to 9.2 U/mL in Stage 5. Catalase levels show a gradual decline from 50.1 U/mL (Stage 1) to 36.8 U/mL (Stage 5). Glutathione Peroxidase (GPx) Levels drop from 10.2 U/mL in Stage 1 to 6.8 U/mL in Stage 5.

Table 5: Association Between Antioxidant Status and CKD Stage

1. Negative Correlations with CKD Progression:

• All biomarkers (SOD, Catalase, GPx, and TAC) show negative correlations, indicating that as CKD progresses, these antioxidant levels decline.
• The strongest negative correlation is observed with Total Antioxidant Capacity (TAC) (-0.52, p < 0.001), suggesting that overall antioxidant defense significantly diminishes as CKD worsens.
• Glutathione Peroxidase (GPx) (-0.48, p < 0.001) also exhibits a strong inverse relationship with CKD severity.

2. Statistical Significance (p-values < 0.001):

• All correlations are highly statistically significant, indicating that these relationships are not due to chance.

Table 6: Multivariate Regression Analysis for Predictors of Microalbuminuria

Negative Predictors (Blue Bars) – Declining Biomarkers with CKD Progression

• Total Antioxidant Capacity (TAC) (-0.35, p < 0.001) → Strongest negative predictor, suggesting that as CKD progresses, TAC declines significantly, increasing oxidative stress.
• Glutathione Peroxidase (GPx) (-0.30, p = 0.002) → Another important antioxidant enzyme showing a strong inverse relationship with CKD.
• Superoxide Dismutase (SOD) (-0.25, p = 0.01) & Catalase (-0.20, p = 0.03) → Moderate negative predictors, further confirming the role of oxidative stress in CKD deterioration.

2. Positive Predictors (Red Bars) – Risk Factors for CKD Progression

• CKD Stage (0.60, p < 0.001) → Strongest positive predictor, meaning that as CKD advances, disease severity correlates significantly with disease progression.
• Diabetes Mellitus (0.25, p = 0.01) → Significant predictor, indicating that diabetes exacerbates CKD by increasing metabolic stress and kidney damage.
• Age (0.10, p = 0.15) → Not statistically significant (p > 0.05), suggesting that while aging may contribute to CKD, it is not a primary driver of disease progression.

Table 7: Comparison of Antioxidant Levels in Patients With and Without Diabetes

• Superoxide Dismutase (SOD):

• Diabetic patients: 10.5 ± 2.8 U/mL
• Non-diabetic patients: 13.2 ± 3.1 U/mL
• p = 0.001 (Statistically significant)

• Catalase:

• Diabetic patients: 42.3 ± 9.5 U/mL
• Non-diabetic patients: 48.2 ± 10.1 U/mL
• p = 0.005 (Statistically significant)

• Glutathione Peroxidase (GPx):

• Diabetic patients: 7.8 ± 1.9 U/mL
• Non-diabetic patients: 9.5 ± 2.0 U/mL
• p = 0.002 (Statistically significant)

• Total Antioxidant Capacity (TAC):

• Diabetic patients: 1.0 ± 0.2 mmol/L
• Non-diabetic patients: 1.3 ± 0.3 mmol/L
• p < 0.001 (Highly significant)

The findings of this study demonstrate a significant negative correlation between antioxidant status and microalbuminuria in chronic kidney disease (CKD) patients, consistent with previous research. Below, we elaborate on these findings in the context of existing literature, explore their implications, and provide a more detailed discussion of the mechanisms and clinical relevance.

1. Antioxidant Status in CKD Patients

Our study revealed that CKD patients had significantly lower levels of antioxidant biomarkers, including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and total antioxidant capacity (TAC), compared to normal ranges. This finding aligns with a robust body of evidence suggesting that oxidative stress is a key pathological feature of CKD. ^[12]

Mechanisms of Oxidative Stress in CKD is characterized by a chronic inflammatory state and increased production of reactive oxygen species (ROS) due to factors such as uremic toxins, hypertension, and dyslipidemia. These ROS overwhelm the body's antioxidant defenses, leading to oxidative damage to lipids, proteins, and DNA. ^[13-17]

Vaziri et al. (2016) highlighted that CKD patients exhibit reduced activity of antioxidant enzymes such as SOD and GPx due to both decreased synthesis and increased degradation. ^[18] Himmelfarb et al. (2002) emphasized that oxidative stress in CKD is exacerbated by the accumulation of advanced glycation end products (AGEs) and pro-inflammatory cytokines, which further deplete antioxidant reserves. ^[19]

Our study observed a progressive decline in antioxidant levels across CKD stages, with the most pronounced reductions in stages 4 and 5. This is consistent with: Small et al. (2012), who reported that antioxidant enzyme activity decreases as kidney function declines, leading to a vicious cycle of oxidative stress and tissue injury. Cachofeiro et al. (2008), who demonstrated that oxidative stress contributes to renal fibrosis, inflammation, and apoptosis, accelerating CKD progression. ^[20] These findings underscore the importance of monitoring antioxidant status in CKD patients and exploring therapeutic strategies to enhance antioxidant defenses. ^[21]

The progressive decline in antioxidant levels across CKD stages observed in our study is consistent with previous research. Mechanisms Linking Oxidative Stress to CKD Progression through multiple pathways, including: Activation of pro-inflammatory signaling pathways (e.g., NF-κB and TGF-β). Induction of renal fibrosis and tubular atrophy. Promotion of endothelial dysfunction and vascular calcification. Small et al. (2012) reported that oxidative stress accelerates CKD progression by promoting inflammation and fibrosis. ^[22]

Therapeutic Implications the decline in antioxidant levels with CKD progression suggests that antioxidant therapies could potentially slow disease progression. Tbahriti et al. (2013) demonstrated that antioxidant supplementation reduced oxidative stress and improved kidney function in CKD patients. ^[23] Cachofeiro et al. (2008) suggested that targeting oxidative stress could mitigate inflammation and fibrosis, thereby slowing CKD progression. ^[24] These findings highlight the potential of antioxidant therapies as adjunctive treatments for CKD.

This study demonstrates a significant negative correlation between antioxidant status and microalbuminuria in CKD patients, consistent with previous research. The findings highlight the role of oxidative stress in CKD progression and suggest that interventions targeting oxidative stress may help reduce microalbuminuria and slow disease progression. Further research is needed to explore the potential benefits of antioxidant therapies in CKD management

1. Abbasi, M. A., Chertow, G. M., & Hall, Y. N. (2016). End-stage renal disease. New England Journal of Medicine, 375(18), 1775-1787. https://doi.org/10.1056/NEJMra1600825
2. Agarwal, R. (2005). Proinflammatory effects of oxidative stress in chronic kidney disease: Role of additional angiotensin II blockade. American Journal of Physiology-Renal Physiology, 288(5), F813-F822. https://doi.org/10.1152/ajprenal.00324.2004
3. Arici, M., & Walls, J. (2001). End-stage renal disease, atherosclerosis, and cardiovascular mortality: Is C-reactive protein the missing link? Kidney International, 59(2), 407-414. https://doi.org/10.1046/j.1523-1755.2001.059002407.x
4. Baynes, J. W., & Thorpe, S. R. (1999). Role of oxidative stress in diabetic complications: A new perspective on an old paradigm. Diabetes, 48(1), 1-9. https://doi.org/10.2337/diabetes.48.1.1
5. Block, G. A., Klassen, P. S., Lazarus, J. M., Ofsthun, N., Lowrie, E. G., & Chertow, G. M. (2004). Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. Journal of the American Society of Nephrology, 15(8), 2208-2218. https://doi.org/10.1097/01.ASN.0000133041.27682.A2
6. Bonventre, J. V., & Yang, L. (2011). Cellular pathophysiology of ischemic acute kidney injury. Journal of Clinical Investigation, 121(11), 4210-4221. https://doi.org/10.1172/JCI45161
7. Cachofeiro, V., Goicochea, M., de Vinuesa, S. G., Oubiña, P., Lahera, V., & Luño, J. (2008). Oxidative stress and inflammation, a link between chronic kidney disease and cardiovascular disease. Kidney International, 74(S111), S4-S9. https://doi.org/10.1038/ki.2008.516
8. Chen, J., Muntner, P., Hamm, L. L., Jones, D. W., Batuman, V., Fonseca, V., ... & He, J. (2004). The metabolic syndrome and chronic kidney disease in US adults. Annals of Internal Medicine, 140(3), 167-174. https://doi.org/10.7326/0003-4819-140-3-200402030-00007
9. Dounousi, E., Papavasiliou, E., Makedou, A., Ioannou, K., Katopodis, K. P., Tselepis, A., ... & Siamopoulos, K. C. (2006). Oxidative stress is progressively enhanced with advancing stages of CKD. American Journal of Kidney Diseases, 48(5), 752-760. https://doi.org/10.1053/j.ajkd.2006.08.015
10. Dröge, W. (2002). Free radicals in the physiological control of cell function. Physiological Reviews, 82(1), 47-95. https://doi.org/10.1152/physrev.00018.2001
11. Eknoyan, G., Lameire, N., Eckardt, K. U., Kasiske, B. L., Wheeler, D. C., & Levin, A. (2013). KDIGO 2012 clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney International Supplements, 3(1), 1-150. https://doi.org/10.1038/kisup.2012.73
12. Forbes, J. M., Coughlan, M. T., & Cooper, M. E. (2008). Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes, 57(6), 1446-1454. https://doi.org/10.2337/db08-0057
13. Giugliano, D., Ceriello, A., & Paolisso, G. (1996). Oxidative stress and diabetic vascular complications. Diabetes Care, 19(3), 257-267. https://doi.org/10.2337/diacare.19.3.257
14. Go, A. S., Chertow, G. M., Fan, D., McCulloch, C. E., & Hsu, C. Y. (2004). Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. New England Journal of Medicine, 351(13), 1296-1305. https://doi.org/10.1056/NEJMoa041031
15. Halliwell, B., & Gutteridge, J. M. C. (2015). Free radicals in biology and medicine. Oxford University Press. https://doi.org/10.1093/acprof:oso/9780198717478.001.0001
16. Himmelfarb, J., Stenvinkel, P., Ikizler, T. A., & Hakim, R. M. (2002). The elephant in uremia: Oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney International, 62(5), 1524-1538. https://doi.org/10.1046/j.1523-1755.2002.00600.x
17. Jha, V., Garcia-Garcia, G., Iseki, K., Li, Z., Naicker, S., Plattner, B., ... & Yang, C. W. (2013). Chronic kidney disease: Global dimension and perspectives. The Lancet, 382(9888), 260-272. https://doi.org/10.1016/S0140-6736(13)60687-X
18. (2002). Clinical practice guidelines for chronic kidney disease: Evaluation, classification, and stratification. American Journal of Kidney Diseases, 39(2 Suppl 1), S1-S266. https://doi.org/10.1053/ajkd.2002.30944
19. Levey, A. S., Coresh, J., Balk, E., Kausz, A. T., Levin, A., Steffes, M. W., ... & Eknoyan, G. (2003). National Kidney Foundation practice guidelines for chronic kidney disease: Evaluation, classification, and stratification. Annals of Internal Medicine, 139(2), 137-147. https://doi.org/10.7326/0003-4819-139-2-200307150-00013
20. Locatelli, F., Canaud, B., Eckardt, K. U., Stenvinkel, P., Wanner, C., & Zoccali, C. (2003). Oxidative stress in end-stage renal disease: An emerging threat to patient outcome. Nephrology Dialysis Transplantation, 18(7), 1272-1280. https://doi.org/10.1093/ndt/gfg074
21. Massy, Z. A., & Drüeke, T. B. (2012). Oxidative stress and chronic kidney disease. Oxidative Stress in Applied Basic Research and Clinical Practice, 1-20. https://doi.org/10.1007/978-1-61779-458-2_1
22. Muntner, P., He, J., Hamm, L., Loria, C., & Whelton, P. K. (2002). Renal insufficiency and subsequent death resulting from cardiovascular disease in the United States. Journal of the American Society of Nephrology, 13(3), 745-753. https://doi.org/10.1681/ASN.V133745
23. Nistala, R., & Whaley-Connell, A. (2012). Oxidative stress and cardiovascular risk in chronic kidney disease. Journal of the American Society of Hypertension, 6(6), 427-434. https://doi.org/10.1016/j.jash.2012.08.003
24. Oberg, B. P., McMenamin, E., Lucas, F. L., McMonagle, E., Morrow, J., Ikizler, T. A., & Himmelfarb, J. (2004). Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney International, 65(3), 1009-1016. https://doi.org/10.1111/j.1523-1755.2004.00465.x