The world’s clinical diagnostic laboratories (CDLs) are gradually moving toward a personalized medicine [1], which represents a modern approach to health protection considering specific features of each patient, including genetic markers and variable phenotypic traits. Such an approach makes it possible to more accurately diagnose the disease, to select optimal treatment and prevention methods, to reduce the risk of complications [2]. Introduction of the personalized medicine and specialized CDLs will enable improving the quality of medical care, reducing the costs of public health services, and improving the patients’ prognosis [3].

When discussing personalized medicine, we cannot ignore a disorder that is called the non-infectious pandemic of the 20th and 21^st centuries. In 2022, there were almost 9 million people suffering from diabetes mellitus (DM) all over the world [4]. Furthermore, the increase in the number of individuals suffering from both type 2 and type 1 DM is reported [5]. It is possible to prevent type 2 DM or alleviate its course and complications through adjustment of lifestyle and diet, increase in physical activity. However, we still cannot affect the development of type 1 DM. Cardiovascular disorders, the risk of which increases 2–4-fold after the onset of diabetic nephropathy (DN), represent the main cause of mortality among patients with type 1 and type 2 DM [6, 7]. DN is a terrible complication of DM, because clinical symptoms manifest themselves at the latest stages only. According to the research, the earlystage chronic kidney disease (CKD) is missed in 20% of patients [8]. DN in patients with type 1 DM manifests itself within 10 years after the disease onset on average. In case of DM decompensation, this median is shifted, and the CKD progression takes place earlier [9]. In patients with type 2 DM, it is recommended to estimate DN severity immediately when making the diagnosis of DM. This is associated with the fact that due to mild manifestation of type 2 diabetes, patients can have hyperglycemia for a long time, which adversely affects the renal function. Inability to use modern nephroprotective drugs (sodium-glucose cotransporter 2 (SGLT-2) inhibitors; glucagonlike peptide-1 agonists, thiazolidinediones) slowing down the CKD progression is an important problem of patients with type 1 DM. The use of such drugs in patients with type 1 DM has not been sufficiently studied and is currently contraindicated [10]. As for individuals with type 2 DM, the use of nephroprotective drugs, on the contrary, has a beneficial effect on the course of DN, since these drugs maintain the glomerular filtration rate (GFR) and reduce albuminuria [11]. The methods to estimate severity of the diabetic complications, specifically DN, are used in the clinical laboratory diagnosis. However, all these methods (determination of the renal function of filtration through calculation of creatinine levels, albumin-to-creatinine ratio (ACR) in a single urinary specimen, micro- or macroalbuminuria in 24 h urine, cystatin C, etc.) become effective after the CKD onset. There are no clear markers warning physicians about the likelihood of severe diabetes and its complications, specifically DN. In our opinion, the molecular genetic markers can inform the physician about the diabetes severity. It is important to choose the genes capable of affecting the course of DN with high sensitivity and specificity amongst the multitude of genes. The leading diabetologists of our country have described the importance of the search for polygenic associations instead of individual genes and have identified the genes responsible for antioxidant protection as the candidate genes [12]. Introduction of point genetic associations into clinical laboratory diagnosis will allow the physician to determine the risk of severe diabetes and its complications, prevent these complications in a timely manner, intensify the glucose-lowering therapy, increase the number of the patients’ preventive check-ups focusing on prevention and identification of the specific genetically determined complication. This represents a fundamental link of the personalized medicine in general and the personalized clinical laboratory diagnosis in particular.

When performing the search for probable candidate genes involved in the DN pathogenesis, we became interested in the role of the GSTP1 (I105V) gene. This gene encodes the glutathione-S-transferase (GST) enzyme being one of the main contributors to the xenobiotic transformation process. Normally, GST contributes to the glutamate interaction with the electrophilic nitrogen (N), carbon (C), sulfur (S) and oxygen (O) atoms and ensures conjugation of sulfhydryl groups with the xenobiotic molecules. The detoxification process driven by GST is the key process of protecting cells from lipid peroxidation and protein alkylation, which increases the resistance to hypoxic states [13]. The GSTP1 gene I105V (A>G) polymorphism is associated with the adenine (A) nucleotide substitution with guanine (G), which results in substitution of the amino acid in the enzyme peptide chain, thereby reducing the enzyme activity and, consequently, increasing accumulation of free radicals in the body. The increased risk of various forms of lung cancer and oral cancer is observed in the G/G genotype carriers [14]. This polymorphism is also associated with susceptibility to leukemia and Parkinson's disease. There are deletion polymorphisms (GSTM (del), GSTT1) that determine the nonfunctional null alleles. It is assumed that individuals with these deletions in homozygous state have a decreased ability to detoxify chemical substances. Such polymorphisms are most often found in women with endometriosis and individuals with allergy [15]. Recently, it has been found out that there are studies, in which the GSTP1 (I105V) gene affects the drug pharmacokinetics, along with the genes of the P-glycoprotein transporter (MDR1), organic cations (OCT1), and organic anions (OATP-C, OAT1, OAT3) [16].

The study was aimed to identify the molecular genetic markers determining the severity of diabetic nephropathy in patients with type 1 and 2 DM based on the GSTP1 (I105V) gene assessment.

METHODS

The study was conducted at the Department of Endocrinology of the Faculty of Advanced Training and Retraining of Specialists of the Kuban State Medical University. Patients were enrolled at the Regional Clinical Hospital of Emergency Medical Care (Krasnodar). A total of 51 individuals with type 2 DM and 49 individuals with type 1 DM were included in this open-label prospective cohort study. Inclusion criteria: patients’ age 20–60 years; type 1 and 2 DM duration 10–15 years; glycated hemoglobin levels of 7.0%–9.5%; glomerular filtration rate exceeding 45 mL/min/1.73 m², regardless of the 24 h urinary albumin excretion rate; using drugs having no nephroprotective effects (biguanides; sulfonylureas; dipeptidyl peptidase-4 inhibitors; insulin) as glucose-lowering therapy; no severe comorbidities at the time of the study. Individuals, who did not meet these criteria, were excluded from the study. We also formed the control group of 20 conditionally healthy donors (13 females and 7 males), who were not related to the index group patients and had no history of DM or kidney disease.

Clinical laboratory testing involved the serum samples obtained by centrifuging the whole blood-containing tubes at 3000 rpm and room temperature. The fasting blood glucose levels, postprandial glycemia, complete blood counts, and blood biochemistry were assessed using the Konelab analyzer (Thermo Fisher Scientific; Finland); urinalysis and 24-h urine tests were performed using the SYNCHRON СХ9 PRO biochemical analyzer (Beckman Coulter; USA) by immunoturbidimetry. GFR (mL/min/1.73 m²) was estimated by calculation using the CKD-EPI formula.

The balance of the pro-/antioxidant system of surveyed patients and the control group was assessed based on the activity of the antioxidant defense system enzymes (superoxide dismutase (SOD) [17], catalase (CAT) [18], glutathione-Stransferase (GSТ) [19] and malondialdehyde (MDA) levels [20]). SOD activity was determined based on the ability to inhibit autoxidation of epinephrine in the alkaline environment. The reaction rate was measured by spectrophotometry based on the resulting absorbance of the released epinephrine autoxidation products in the test sample and relative to the data obtained when there was no epinephrine in the studied blood sample. CAT measurement in the hemolysate was performed by photometry based on the ability to disrupt Н₂О₂. The essence of the GSТ determination method was the ability of reduced glutathione (present in the substrate — 1% hemolysate of patient’s red blood cells) to bind 1-chloro-2,4dinitrobenzene, forming a stable chromogenic conjugate in the alkaline environment. The qualitative assay used to estimate MDA levels in the hemolysate involved adding thiobarbituric acid in the presence of chloroacetic acid.

The GSTP1 (I105V) molecular genetic testing was performed in the molecular genetic research laboratory of the Kuban State Medical University (Krasnodar). The real-time polymerase chain reaction (qPCR) and the RotorGene real-time PCR cycler (QIAGEN; Germany) were used to perform genotyping of the GSTP1 gene I105V locus from the leukocyte fraction. Two amplification reactions with the extracted DNA sample were simultaneously launched (with two pairs of allele-specific primers). The cycler automatically detected the amplification products in each amplification cycle. According to the data obtained, the control program plotted the fluorescent signal accumulation curves for the channel specified for the sample. The analysis results allowed us to draw conclusions of three types: homozygote for allele 1; heterozygote; homozygote for allele 2.

Statistical analysis

Significance of differences in the genotype frequency distribution between the groups of patients with DM and healthy individuals was assessed using the χ² test, and the quantitative indicators of the patients’ clinical characteristics were assessed using the Student’s t-test. Calculations were performed using the BIOSTAT software. The differences were considered significant at p < 0.05. To determine compliance with the Hardy–Weinberg principle, we calculated frequencies of all allele variants and the corresponding χ² values. The critical χ² value exceeded the estimates calculated for each group, suggesting that the Hardy–Weinberg equilibrium was preserved.

RESULTS

The following percentages of the GSTP1 (I105V) polymorphism carriers were revealed among patients with type 2 DM: 25.5% were heterozygous carriers (ILE/VAL), 62.7% were carriers homozygous for allele 1 (ILE/ILE), and 11.8% were carriers homozygous for allele 2 (VAL/VAL). Among patients with type 1 DM, the share of patients with heterozygous gene variant (ILE/VAL) was 34.7%; 65.3% were homozygous for allele 1 (ILE/ILE); no patients homozygous for allele 2 were revealed. These data are significantly different from that of the control group, where 65% were heterozygous carriers (ILE/VAL), 35% were homozygous carriers (ILE/ILE), and where there were no carriers homozygous for allele 2 (VAL/VAL) (tab. 1).

Then we launched the study of the relationship between the renal function and the studied gene polymorphism variant. No differences in GFR and 24 h urinary albumin excretion rate between the heterozygous carriers (ILE/VAL) and the carriers homozygous for the GSTP1 allele 1 (ILE/ILE) were revealed among patients with type 2 DM. The average GFR in the subgroups of individuals with type 2 DM having homozygous and heterozygous polymorphisms was 64 mL/min/1.73 m², and the average protein level in the 24 h urine was 0.18 g/L. However, patients with type 2 DM, who were carriers of the rare homozygote for allele 2 (VAL/VAL), had much worse indicators: the average GFR of this subgroup was 48 mL/min/1.73 m², and the 24 h urinary albumin excretion rate was 0.9 g/L. There were no significant differences in other blood biochemistry indicators between the studied gene allele variants in individuals with type 2 DM.

Assessment of the same indicators in the homo- and heterozygous carriers of the GSTP1 (Ile105Val) polymorphisms in the group of patients with type 1 DM revealed no significant differences in the renal function indicators (GFR and 24 h urinary albumin excretion rate). However, a significant (1.6-fold) increase in the levels of triglycerides and a 1.1-fold increase in the glycated hemoglobin levels in the heterozygous carriers (ILE/VAL) of the gene compared to homozygous carriers (ILE/ILE) were revealed (р < 0.05) (tab. 2).

No comparison of renal function between patients with type 1 DM, patients with type 2 DM, and the controls was performed, since the control group consisted of healthy individuals.

In the next phase we proceeded to assessing the activity of the antioxidant defense (AOD) enzymes and MDA levels as a function of the studied gene polymorphism. The most significant increase in the activity of the AOD enzymes (catalase and superoxide dismutase) among patients with type 2 DM was reported for the carriers homozygous for allele 1 compared to heterozygous carriers of the GSTP1 polymorphisms and the carriers homozygous for allele 2. However, the levels of the advanced glycation end products (MDA) in the subgroup of carriers of the rare homozygote for allele 2 (VAL/VAL) were significantly higher (100.5 µmol/L), than in individuals with other polymorphisms (р < 0.001) [21].

Among patients with type 1 DM, heterozygous carriers of the GSTP1 polymorphism (ILE/VAL) had higher levels of MDA, GST, and catalase compared to homozygous carriers of the gene polymorphism (р < 0.001, р < 0.001, р < 0.05).

Comparison of the data obtained between patients with type 1 DM, patients with type 2 DM, and the controls revealed, quite naturally, a significant predominance of activity of the AOD enzymes and MDA levels in individuals with DM (tab. 3).

DISCUSSION

The study conducted makes it possible to conclude that the abundance of heterozygous GSTP1 polymorphism (ILE/VAL) in individuals with type 1 and 2 DM is significantly higher than that in controls. The genotype distributions for both polymorphisms were compliant with the Hardy–Weinberg equilibrium and showed no significant differences from the data of the SNP database [22]. Furthermore, the rare Val/Val genotype was revealed in individuals with type 2 DM, which was found in none of the surveyed patients with type 1 DM and controls (χ²  = 6.572, р = 0.039).

Patients with type 2 DM, carriers of the rare GSTP1 polymorphic variant homozygous for allele 2 (VAL/VAL), had higher levels of MDA (free radical oxidation markers) and higher activity of the oxidative stress enzymes. One of the studies has shown a clear correlation between this polymorphic variant and the development of type 2 DM, however, the impact of this variant on the development of such complication, as diabetic polyneuropathy, has not been proven [23]. In our study, patients with the rare homozygote for allele 2 had a significantly more severe course of DN (average GFR — 48 mL/min/1.73 m²; average 24 h urinary albumin excretion rate — 0.9 g/L). Perhaps, this is due to the fact that this specific genetic variant causes the decrease in activity of the protein produced (GST), which does not ensure adequate detoxification of xenobiotics and results in the increase in activity of free radical oxidation. This is reflected in the increase in the free radical oxidation marker (MDA) levels. Thus, conditions are being created for active lipid peroxidation in all cells, especially in the cells most sensitive to hypoxic damage, the nephrons. The multifaceted damage to the nephron structure results in the decrease in the renal functions of filtration (elevated creatinine levels and reduced GFR) and reabsorption (elevated 24 h urinary albumin excretion rate) in the cohort of patients with type 2 DM. Similar data were obtained in the study, during which the researchers confirmed the correlation between the rare homozygous polymorphism (VAL/VAL) and the end-stage kidney disease [24].

Heterozygous (ILE/VAL) carriers of the GSTP1 (I105V) gene with type 1 DM had significantly higher activity of the AOD enzymes and MDA levels compared to the homozygous polymorphisms. GST, the antioxidant enzyme, protects the tissues against oxidative damage typical for many health conditions, especially those like type 1 DM and its chronic complications. This polymorphism is likely to be more pathogenic, when it is in heterozygous state. Similar results were obtained in the study that revealed the correlation between this genetic polymorphism and the development of the diabetic complication (cardiovascular autonomic neuropathy) in patients with type 1 DM [25]. Patients with type 1 DM and the heterozygous polymorphism variant also demonstrate a significant increase in the levels of triglycerides (1.6-fold). Based on these data, we can assume that heterozygous carriers have the reduced triacylglycerol lipase activity, which results in slower breakdown of triglycerides. This is associated with the influence of oxidative stress and some hormones, such as norepinephrine, epinephrine, glucagon, etc. it has been reported that activity of this enzyme can change under the influence of the discussed factors [26]. Furthermore, significantly higher glycated hemoglobin levels were determined in this subgroup of patients, which also correlated with elevated MDA levels and increased activity of CAT and GST.

CONCLUSIONS

The personalized medicine is based on the tailored approach to the characteristics of each patient. Introduction of the GSTP1 (Ile/Val) gene molecular genetic assessment into clinical laboratory diagnosis will make it possible to identify patients with type 1 and 2 DM having high levels of oxidative stress and increased risk of severe DN. When performing clinical laboratory diagnosis, it is recommended to determine the heterozygous (ILE/VAL) variant of GSTP1 (I105V) in individuals with type 1 DM, which is associated with the increased free radical oxidation and activity of the AOD enzymes and can result in the significantly higher glycated hemoglobin and triglyceride levels. As for patients with type 2 DM, it is recommended to determine the rare GSTP1 (I105V) gene variant homozygous for allele 2 (VAL/VAL) that is associated with the increased free radical oxidation and activity of the oxidative stress enzymes, as well as with the reduced renal function (increased 24 h urinary albumin excretion rate and reduced GFR). Such studies can provide the basis for development of the genetic panel, in which the polygenic sequences that improve reliability and ensure more accurate prediction of the severity and timing of the onset of diabetic complications will be determined. However, this requires expansion of the research in this field.