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Erythrocyte Superoxide Dismutase as a Marker for Nephropathy in Type 2 Diabetes Mellitus: A Pilot Study
* Corresponding author: Dr. Shilpa Bhardwaj, Department of Neurochemistry, Institute of Human Behaviour and Allied Sciences, Delhi, India. drshilpa2001@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Bhardwaj S, Singh K, Singh R. Erythrocyte Superoxide Dismutase as a Marker for Nephropathy in Type 2 Diabetes Mellitus: A Pilot Study. Int J Recent Surg Med DOI: 10.25259/IJRSMS_23_2024
Abstract
Objectives
Hyperglycemia, characterized by elevated blood glucose levels, is a consequent upregulation in the generation of reactive oxygen species (ROS). These ROS possess the capacity to induce deleterious modifications in endothelial function. Superoxide dismutase (SOD), an enzymatic antioxidant, stands as the primary scavenger of superoxide, a specific type of ROS, within the extracellular space. This study aims to elucidate the association between the levels of erythrocyte SOD and routinely measured renal function parameters (serum urea and serum creatinine) in type 2 diabetes mellitus (T2DM) patients with nephropathy.
Material and Methods
Diagnosed patients of T2DM were enrolled in the study conducted at a tertiary care hospital in Delhi, India (n = 120). Erythrocyte SOD concentrations (expressed as units/g of hemoglobin) were determined in 60 T2DM patients with nephropathy (cases) and 60 type 2 diabetes patients without any complications (controls) using a commercially available colorimetric assay kit on an automated analyzer. Serum urea and creatinine levels were measured spectrophotometrically on a clinical chemistry automated analyzer. Data were analyzed using SPSS Statistics (v.20) for statistical inference.
Results
We observed significantly lower activity of erythrocyte SOD with mean levels of 183 ± 0.78 units/g hemoglobin in subjects with DM nephropathy compared with mean levels of 186 ± 0.95 units/g hemoglobin in the control subjects (P = 0.03). Our analysis revealed a statistically significant negative association (p-value = 0.02) between erythrocyte SOD activity and serum creatinine levels in DM patients diagnosed with nephropathy.
Conclusion
Our findings suggest that lower levels of erythrocyte SOD are associated with nephropathy in T2DM.
Keywords
Diabetes mellitus
Nephropathy
Superoxide dismutase
INTRODUCTION
Diabetes mellitus (DM) represents a chronic, multifactorial metabolic disorder characterized by persistent elevations in blood glucose (hyperglycemia). The globally rising epidemic of DM has not spared the developing countries, with approximately 70 million people expected to be affected by 2025.[1] Oxidative stress is a well-known precursor of progressive vascular damage and the eventual complications seen in DM. Endothelial dysfunction, characterized by an imbalance between the markers of oxidative stress and various antioxidants, plays a crucial role in initiating vascular injury and inflammation in diabetes. This chronic vascular insult results in progressive damage to kidneys, and once nephropathy ensues, measures can only be taken to halt its progression. Currently, DM nephropathy represents the predominant aetiology contributing to both morbidity and mortality in the context of chronic diabetic complications. Several prospective studies have established a link between high blood glucose levels and cardiovascular morbidity in diabetes.[2] Several mechanisms have been postulated for hyperglycemia-mediated damage, such as cytotoxicity, enhanced extracellular matrix production, vascular dysfunction, and oxidative stress.[3] Persistent hyperglycemia due to defective insulin secretion or insulin insensitivity leads to the generation of ROS species from glucose autoxidation and protein glycosylation.[4] Free radicals generated during normal metabolism are scavenged by endogenous enzymatic antioxidants like superoxide dismutase (SOD). In DM, an excess of free radicals is generated, or their destruction by radical scavenging systems is reduced. This imbalance results in vascular injury and predisposes to various complications like DM nephropathy. Recent studies have suggested that elevated blood glucose levels trigger the increased production of superoxide anions within the mitochondrial electron transport chain (ETC). It is the prime step in the pathogenesis of hyperglycemia-induced complications in DM.[5] Superoxide mediates further damage via activation of other pathways, resulting in the production of peroxynitrites, which are even stronger oxidants.[6] These free radicals induce DNA damage to modify the expression of glucose transporter type 4 (GLUT4) in adipocytes and muscle, impair the production and secretion of insulin from β cells in the pancreas, and induce endothelial dysfunction.[7]
SOD is a ubiquitous enzymatic class with the function of catalyzing the dismutation of superoxide anions. In eukaryotic cells, there is a spatial distribution of SOD isoforms: CuZnSOD is predominantly localized within the cytosol, while MnSOD is found primarily within the mitochondrial matrix. The enzymatic action of SOD degrades superoxide ions into peroxides, which are rapidly broken down into water and oxygen by other enzymes. A functional polymorphism in the SOD gene has been linked with type 2 DM in Japanese and Korean populations.[8,9] This investigation aimed to elucidate a potential association of erythrocyte SOD with type 2 DM nephropathy in the North Indian population.
MATERIAL AND METHODS
Selection of subjects
This hospital-based case-control study was conducted at a tertiary care hospital in Delhi by recruiting subjects from among the patients attending the diabetes clinic at a tertiary care hospital in Delhi, India. The case group was formed of 60 patients (age group, 40–70 years old) diagnosed with type 2 diabetes mellitus with nephropathy (based on American Diabetes Association, 2011 criteria). In the context of type 2 DM, nephropathy is diagnosed by either persistent albuminuria exceeding 300 mg/day or 200 μg/min measured on at least two separate occasions three to six months apart or a progressive reduction in glomerular filtration rate (GFR). The control group was comprised of type 2 DM patients without any microvascular or macrovascular complications (n = 60). None of the patients were on insulin treatment.
Exclusion criteria
Pregnant and nursing women and subjects with dehydration; muscular dystrophy; rhabdomyolysis; glomerulonephritis; pyelonephritis; urinary tract infections; severe neurological, endocrinological, dermatological, acute or chronic debilitating illness; end-stage renal disease, or on dialysis were screened out.
Informed written consent was obtained from all participants, and data was obtained using a standard questionnaire prepared with information on age, sex, duration and family history of DM. Anthropometric indicators like blood pressure and body mass index (kg/mt 2) were measured in the morning after taking fasting venous samples. All biochemical analyses were carried out in the clinical biochemistry laboratory of a tertiary care hospital on automated analyzers using standard kits and protocols. All analyses were run in duplicates, and average values were used.
Sample collection
A 5-mL fasting venous sample was collected under sterile conditions from the median cubital vein and immediately processed for separation of plasma (NaF and EDTA vacutainer) for the estimation of glucose and serum (no anticoagulant vacutainer) for urea and creatinine on an automated analyzer (CX series; Beckman Coulter, Inc., Brea, California, USA). Whole blood lysate was employed for the quantification of erythrocytic SOD activity on an automated analyzer platform.
Biochemical assays
Fasting plasma glucose
The concentration of glucose in plasma was determined using a commercial enzymatic assay kit employing the glucose oxidase-peroxidase (GOD-POD) method. 5µL of samples and standard solution (100 mg/dL) were incubated with a reaction mixture for 5 minutes at 37 °C. The reaction mixture comprised 100 mM phosphate buffer at pH 7.5, supplemented with 5 mM phenol, 10 U/mL glucose oxidase, 1 U/mL peroxidase and 0.4 mM 4-aminoantipyrine. Following incubation, a spectrophotometric analysis was performed on the solution at 505 nm using an automated instrument to quantify absorption.
Serum urea estimation
Urease-GLDH, a kinetic UV-based spectroscopic technique was employed for measuring serum urea levels. A working reagent of urea was prepared by dissolving 1 tablet of reagent R.2 (urease 3750 U/L, glutamate dehydrogenase 6000 U/L, NADH 0.32 mmol/L) in 20 mL of R.1 (Tris pH 7.8, α- ketoglutarate). Ten microliters of sample and standard (50 mg/dL) were incubated at 37°C with a working reagent. Absorbance was read after 30 seconds (A1) and 90 seconds (A2) at 340 nm.
Serum creatinine determination
The alkaline picrate kinetic method was used for measuring levels of serum creatinine. A working solution of creatinine reagent solution was prepared by combining equal volumes of solution R1a (picric acid, 35 mmol/L) and R1b (sodium hydroxide, 0.32 mol/L). Ten microlitres of samples and standards were incubated with 100 μL of working reagent, and absorbance was recorded at 490 nm.
SOD; E.C.1.15.1.1 activity estimation
After carefully sampling erythrocytes from the bottom of the tubes, washing was done thrice with isotonic saline solution. Double-distilled water containing 5 mL/L Triton X-100 was added to lyse the erythrocytes and stored on ice for 10 minutes following vortex mixing. SOD activity in erythrocytes was quantified using commercially available RANSOD kits (cat. No. SD 125; Randox Labs., Crumlin, Northern Ireland). This assay utilizes xanthine oxidase, which generates superoxide radicals by acting on its substrate, xanthine. The superoxide radicals then interact with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT), leading to the formation of a red-coloured formazan dye. SOD activity is quantified by its capacity to inhibit the rate of a specific reaction. Absorbance was measured at 505 nm on an automated analyzer. SOD activity is quantified in whole blood (initial state) as units per milliliter. One unit signifies the enzyme quantity necessary to achieve a 50% reduction in formazan formation rate. This was then converted to units per gram of hemoglobin (specific activity). A standard curve was plotted using standards by serial dilutions of a commercially available SOD calibrator along with the kit.
Statistical analyses
This study utilized SPSS Statistics version 20 (SPSS, Inc., Chicago, IL) for data analysis. Data are presented as mean ± SEM. Independent t-tests assessed intergroup differences in quantitative measures. Pearson correlation coefficients assessed the strength and direction of the linear relationship between study variables. Regression analysis was conducted to identify the variable with the most significant predictive power for nephropathy in type 2 DM. A p-value ≤ 0.05 was considered statistically significant.
RESULTS
Based on the presence of diabetic nephropathy, the study population was categorized into two distinct groups for further analysis. The case group comprised individuals diagnosed with T2DM and concomitant nephropathy (n = 60). The control group consisted of T2DM patients without any complications (n = 60). Demographic and disease-related characteristics of the participants at baseline are presented in Table 1 to facilitate group comparisons. Statistical analysis revealed no significant discrepancies between the groups’ mean age and diabetes duration. Biochemical analysis results have been depicted in Table 1. Fasting plasma glucose levels were not markedly different between the two groups, even though the values were on the higher side in cases compared to controls (P = 0.07). Serum urea (P = 0.02) and serum creatinine (P = 0.002) levels were significantly different with mean levels higher in cases compared to controls. Erythrocyte SOD activity, measured in units per gram of hemoglobin, was significantly lower in subjects with type 2 DM and nephropathy compared to those with type 2 DM alone (p-value = 0.03). Correlation analysis revealed a statistically significant negative association between serum creatinine concentration and SOD (SOD) activity. The coefficient of –0.337 suggests a moderate negative relationship, indicating that higher levels of creatinine are associated with lower SOD activity. This finding was statistically significant at a p-value less than (p < 0.05), as detailed in Table 2. Multivariate regression analysis using SOD as a dependent variable showed its significant predictability for DM nephropathy with variations in serum creatinine (P = 0.01), as shown in Table 3.
| Characteristics | Controls mean ± SEM | Cases mean ± SEM | P value |
|---|---|---|---|
| Age (years) | 58.42 ± 1.12 | 58.96 ± 1.02 | 0.14 |
| Duration of diabetes (years) | 5.82 ± 0.64 | 5.94 ± 0.56 | 0.12 |
| Fasting plasma glucose (mg/dL) | 126 ± 2.64 | 132 ± 3.2 | 0.07 |
| Serum urea (mg/dL) | 26 ± 1.0 | 31.2 ± 1.8 | 0.02* |
| Serum creatinine (mg/dL) | 0.9 ± 0.02 | 1.1 ± 0.05 | 0.002* |
| Erythrocyte SOD activity (units/g hemoglobin) | 186 ± 0.78 | 183 ± 0.95 | 0.03* |
SOD: Superoxide dismutase, SEM: Standard error of mean
| Parameter | Pearson coefficient “r” | P value |
|---|---|---|
| Serum urea | –0.07 | 0.64 |
| Serum creatinine | –0.337 | 0.02* |
| Parameter | Beta value (standardized coefficient) | P value | 95% Confidence Interval |
|---|---|---|---|
| Serum urea | 0.174 | 0.28 | –0.08 to 0.259 |
| Serum creatinine | –0.433 | 0.01* | –13.5 to –1.9 |
DISCUSSION
Nephropathy is a common and debilitating complication of type 2 DM, which drastically impairs quality of life. Once diagnosed, its progress is difficult to control, and a complete cure is hard to achieve, but in developing stages, its progression can be controlled. We conducted this study to establish the association of erythrocyte SOD with type 2 DM nephropathy. We tried to evaluate the role of erythrocytic SOD as an early biomarker in a scenario where urinary markers are presently considered superior to serum markers for diagnosing and monitoring the progression of the disease, keeping in mind that the feasibility of procuring an ideal urine sample for analysis has more limitations, including refrigeration, contamination, and cumbersome collection, as compared to a blood sample.[10]
This investigation focused on T2DM patients with nephropathy who had significantly lower levels of erythrocyte SOD activity as compared to their respective controls (P = 0.03). Our results were comparable to the previous studies, which also reported decreased SOD activity in DM nephropathy.[11] SOD scavenges the superoxide reactive oxygen species (ROS), which are formed in the mitochondria during prolonged hyperglycemia.[12] A long-term prospective study over 20 years showed that nephropathy with the fast track is associated with enhanced mitochondrial ROS production in DM patients as compared to slow-track nephropathy in DM.[13] Shen et al.[13] suggested a protective role of increased expression of SOD in reducing DM cardiomyopathy.[14] A recent study showed decreased activity of renal SOD in the pathogenesis of nephropathy in mouse models with diabetes.[15]
We also observed significantly higher levels of biochemical indicators of renal function (i.e., serum urea [P = 0.02] and creatinine [P = 0.002]) in DM patients with nephropathy as compared to controls. Idonije et al. reported similar findings in type 2 DM patients with nephropathy.[15] These two serum metabolites have been used as predictors of nephropathy in diabetic patients as they are excreted through the kidneys. Serum creatinine is considered a better marker as it is largely excreted and not reabsorbed by the kidneys and it is not affected by diet, age and other factors that affect serum urea levels. Creatinine clearance is used as an estimate of the glomerular filtration rate, which declines as renal function is impaired in chronic kidney disease. We observed that serum creatinine levels were more significantly associated with nephropathy as compared to serum urea. Siddiqui K reported the use of serum creatinine as a marker for progression of DM nephropathy.[16]
The levels of plasma glucose in the two groups were not significantly different, but the DM patients with nephropathy had higher levels of plasma glucose as compared to DM patients with no complications (P = 0.07). Chronic or uncontrolled hyperglycemia contributes to complications in DM through five major pathways in which the overproduction of reactive oxygen species forms the primary event.[17-19]
In our study, Pearson’s correlation analysis demonstrated a statistically significant negative association between serum creatinine levels and SOD activity within the case group (p = 0.02). This rise in serum creatinine suggests progressive renal damage and decreased glomerular mass, which correlates with the decreased activity of SOD. Studies have suggested that downregulation of SOD in DM patients with nephropathy can be mediated by TNF-α (Tumour necrosis factor- alpha) and other cytokines, thus mitigating the protective effect of SOD in oxidative stress in complicated DM.[20] We also found a negative correlation between serum urea and SOD activity in DM patients with nephropathy, even though it did not reach statistically significant levels.
Multiple regression analysis showed that serum SOD activity (dependent variable) changed significantly with slight changes in serum creatinine levels (independent variable) with β value/standardized coefficient value of –0.433 significant at P = 0.01, keeping the other independent variable, serum urea, as a constant. No significant change was observed in SOD activity variations with serum urea when creatinine was kept as a constant variable (P > 0.05). The rate of change of the conditional mean of serum SOD activity with respect to serum creatinine was expected to be between 1.9 and 13.5 (95% CI) in the negative direction, suggesting a higher predictability of SOD for DM nephropathy.
The present study indicates that the levels of SOD decrease significantly in type 2 DM patients with nephropathy, suggestive of oxidative stress-mediated damage. Thus, the lower erythrocyte SOD levels in patients with type 2 DM may contribute to the progression of renal complications.
Limitations of the study
In this study, we investigated the relationship of the superoxide scavenger, superoxide dismutase, with the most common cause of chronic renal disease, DM nephropathy. The limitations of the study were the confounding factors or other mechanisms which may play a role in the pathogenesis of complications in DM. Moreover, in this study, we could not correlate the levels of erythrocyte SOD with urinary markers (GFR and microalbumin or proteinuria), which are presently considered to be the earliest markers of nephropathy in DM and for which further studies are anticipated. Nevertheless, we could establish a role for SOD as a better predictor than routinely utilized serum indicators (urea and creatinine) in the pathogenesis of DM nephropathy, but for a cause-and-effect relationship, large-scale studies would be required.
CONCLUSION
This case-control study suggested that nephropathy is associated with reduced activity of erythrocyte SOD in type 2 diabetes mellitus due to hyperglycemia-mediated toxic effects, and therefore, the increased oxidative stress may lead to progression of the disease and its associated complications. Since erythrocytic SOD levels can be estimated on a routine basis in clinical labs using automated analyzers, it is an easy alternative to cumbersome urine-based investigations for early diagnosis of DM nephropathy. The role of free radicals cannot be underestimated in other causes of renal failure. As our study focused on the role of erythrocytic SOD in type 2 DM, similar work can be carried out in type 1 DM as hyperglycemia is a common feature. Moreover, inflammation has now been well recognized as one of the mechanisms for CAD and CVD which opens the platform for research of this biomarker to look at their predictability. Large-scale studies would be needed to establish more specific guidelines for this parameter. At present, the role of antioxidants in diabetic patients cannot be denied, and supplementation in the early stages may be beneficial in delaying the development of complications in type 2 diabetes mellitus.
Acknowledgement
The authors would like to thank the patients and the hospital staff, especially technicians, for their cooperation during the study.
Ethical approval
The Institutional Review Board has waived the ethical approval for this study.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
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