Diabetic nephropathy (DN) is the main microangiopathic complication of type 2 diabetes mellitus (DM2). It is a leading cause of chronic kidney disease (CKD) and kidney failure. Diabetic patients are between 25 and 45 percent of all dialysis patients in both Bulgaria and Europe. The group of diabetics who need dialysis is the biggest of all. Many studies mention the use of different biomarkers preceding the development of CKD. Such biomarkers are: biomarkers for genetic, serum, tubular vascular-endothelial dysfunctions.^[1–5]

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which catalyzes the conversion to 5-methyltetrahydrofolate cosubstrate for homocysteine remethylation to methionine.^[6]

MTHFR gene is located in the 1p 36.3 chromosome. Gene polymorphism of MTHFR, C677T (rs1801133), C→T transition at nucleotide 677 in exon 4 is a common gene variant of MTHFR. The most common variations of the polymorphism of MTHFR gene are MTHFR C677C = normal MTHFR gene, MTHFR C677T = heterozygous mutation, MTHFR T677T = homozygous mutation. These genetic polymorphisms can play the role of ‘defects’ which limit the production of the MTHFR enzyme. This mutation causes the amino acid alanine to change into valine. The result is a thermolabile variant of MTHFR with reduced catalytic activity, which can be stabilized by folic acid. The MTHFR T677T homozygous mutations reduce enzyme activity by 50% and are the most common cause of familial moderate hyperhomocysteinemia. This polymorphism determines the catalytic domain of the enzyme and the formation of thermolabile protein. Homocysteine plays a key role in the metabolism of essential amino acids and methionine. Elevated homocysteine levels are identified as a risk factor for DN in DM2. According to Moczulski et al., С677Т polymorphism is a risk factor for DN in men with DM2. They have registered high rate of CT and TT genotypes in men on hemodialysis with DM2 which is in correlation with the presence of С677Т allele and the development of DN according to studies conducted by Japanese researchers.^[7]

Chen et al.^[8], summarizing several meta-analyses based on 13 studies containing 891 healthy people (894 with diabetic nephropathy and 1261 with diabetes mellitus and without DN), assess the association between MTHFR C677T polymorphism and type 2 diabetes mellitus and/or diabetic nephropathy. They found that the 667T allele exhibits significant relation with diabetic nephropathy (p<0.00001), but there is no relation with diabetes mellitus (p=0.25). Movva et al. confirm that MTHFR 677Т may be a risk factor for development of diabetic nephropathy.‌^[9] Benrahma et al. have demonstrated that the MTHFR 677Т polymorphism may be a risk factor for diabetic nephropathy.^[10] Carriers of MTHFR 677T allele are related by a progression of diabetic nephropathy within a period of 5 to 10 years.

Plasminogen activator inhibitor1 (PAI1) is the main inhibitor of fibrinolysis. It is a linear glycoprotein with molecular mass of 48 kDa, which contains 379 amino acids. PAI1 is one of the inhibitors of serine proteinase and is a key regulatory element in fibrinolysis. The main function of PAI1 is to inhibit the activity of the tissue plasminogen activator, which participates in the transformation of plasminogen into plasmin. The increased activity of plasma PAI1 leads to reduced fibrinolytic activity.^[11]

The gene encoding PAI1 is located on the short arm of chromosome 7q 21.3 and contains 9 exons and 8 introns. A polymorphism in the promoter region of PAI1 in the base of 675 from the beginning of the transcription region which refers to 3 or 4 guanine bases (4G/5G). 4G/5G polymorphism has a functional role in determining the base levels of PAI1. Homozygous 4G/4G is related to the transcription of the gene and increased gene expression, which leads to a 25% increase in PAI1 plasma concentration in comparison to 5G/5G genotype. Guanine insertion/deletion polymorphism (4G/5G) in the promoter region of the gene on position 675 regulates PAI1 expression and influences the binding of specific transcription factors and gene transcription rate.

Meigs et al. found that increased plasminogen activator inhibitor-1 plasma levels increase the risk for type 2 diabetes.^[12] PAI1 4G/5G polymorphism is a main genetic determinant of PAI1 plasma levels, 4G/4G homozygotes increase PAI1 level in comparison to 5G allele carriers. These observations suggest the hypothesis that PAI1 4G/5G polymorphism may be a genetic risk factor for diabetes and diabetic nephropathy. Several studies from 2006 and 2012 found that 4G/4G polymorphism of plasminogen activator inhibitor 1 gene polymorphism increases the risk for developing diabetic nephropathy in patients with type 2 diabetes mellitus.^[12–15]

The object of interest is diabetic nephropathy in patients with DM2.

One hundred fifty patients were studied and allocated to three groups. Group 1 consisted of 67 patients with DM2 and DN with stage 1 or 2 CKD (serum creatinine 96.22±18.61 µmol/l, creatinine clearance by MDRD 103.26±16.12 ml/min). Group 2 included 45 patients with DM2 and no clinical and laboratory evidence of DN. Group 3 was the control group and consisted of 38 healthy individuals. Diagnosis of DM2 was based on the WHO criteria.

Inclusion criteria for the first two groups

1. Type 2 diabetes mellitus; 2. Age at the debut of diabetes >18; 3. Duration of DM2 more than 3 years; 4. Glycated hemoglobin (HbA1c) up to 7.0%; 5. No infection for the past three months; 6. Normal or medically controlled blood pressure.

Exclusion criteria for the first two groups

1. Type 1 diabetes mellitus; 2. Type 2 diabetes mellitus with poor metabolic control; 3. High grade proteinuria (>3.0 g/l); 4. Primary kidney disease; 5. Glomerulopathy in lupus or other collagenoses; 6. Primary or secondary amyloidosis; 7. Uncontrolled hypertension; 8. Ischemic heart disease and its complications; 9. Vascular brain disease and its complications; 10. Other macroangiopathic complications of diabetes mellitus; 11. COPD, asthma; 12. Chronic liver disease; 13. Neoplastic processes; 14. Acute or chronic inflammation on active treatment; 15. Chronic alcohol abuse; 16. Continuous use (in the last six months and during the study) of nonsteroidal anti-inflammatory drugs, corticosteroids, hormonal drugs, and antioxidants.

The distribution of the three groups is presented in Table 2. The groups differed in both age and sex. The controls were the youngest, and there was a difference in the mean age between groups 1 and 2. The distribution by sex was similar – the second group was equal to M/F, in group 1 there were more men, and in the control group there were more women (Table 2).

The results for real-time PCR for determining polymorphism C677T of the gene for MTHFR are presented in Table 3. Patients with DM2 and DN with stage 1 and 2 of CKD with a normal genotype were 31.34%, heterozygous С677Т – 47.76%, and homozygous 677Т>Т – 20.90%. Patients with DM2 and no DN with a normal genotype were 46.66%, heterozygous С677Т – 44.44%, and homozygous 677Т>Т – 8.90%. Healthy controls with a normal genotype were 79.0%, heterozygous – С677Т were 13.0%, and homozygous 677Т>Т – 8.0%. (chi-square 23.94, p<0.001) (Table 3).

Furthermore, we have studied the 24-month tendency of the serum creatinine in association with the polymorphism С677Т of the gene for MTHFR in patients with DM2 and DN. In homozygous MTHFR 677T>T serum creatinine progressed (117.18–200.13 µmol/l) much faster than the heterozygous MTHFR 677C >T (116.74–176.63 µmol/l) and those with a normal genotype (113.94–157.05 µmol/l) (p<0.05) (Fig. 1). The analysis showed a very strong correlation between serum creatinine and the C677T polymorphism of the MTHFR – R2 gene is 0.9989 in homozygotes, R2 – 0.9497 in heterozygotes, and R2 – 0.9988 in those with normal genotype (Fig. 1).

We investigated the 24-month trend of the albumin/creatinine ratio in morning urine in relation with polymorphism C677T of the gene for MTHFR in patients with DM2 and DN. In homozygous MTHFR 677T>T albumin/creatinine (0.980–1.56 mg/mmol) progressed much faster in comparison to the heterozygous MTHFR 677C >T (0.362–1.360 mg/mmol) and those with a normal genotype (0.762–1.233 mg/mmol). In heterozygous MTHFR 677C >T albumin/creatinine ratio (0.362–1.360 mg/mmol) in morning urine progressed much slower compared to those with a normal genotype (0.762–1.233 mg/mmol) (p<0.05) (Fig. 2). The analysis showed a very strong correlation between the albumin/creatinine ratio and the C677T polymorphism of the MTHFR – R2 gene is 0.9412 in homozygotes, R2 – 0.9413 in heterozygotes, and R2 – 0.9988 in those with normal genotype (Fig. 2).

High correlation of creatinine clearance in regard to polymorphism C677Т of the MTHFR gene in patients with DM2 and DN is demonstrated – in homozygous MTHFR 677T>T creatinine clearance is the lowest 75.6 ml/min, higher in heterozygous MTHFR 677C>T 108.63 ml/min, and the highest in normal genotype 113 ml/min (Fig. 3). The analysis showed a very strong correlation between creatinine clearance and C677T polymorphism of the MTHFR – R2 gene is 0.8363 (p<0.05) (Fig. 3).

The results for the 4G/5G polymorphism of the gene for PAI-1 in patients with DM2 and DN with stage 1 and 2 of CKD are demonstrated – the ones with a normal genotype were 16.4%, heterozygous 4G/5G were 58.2%, and homozygous 4G/4G were 25.4%. Patients with DM2 and no clinical or laboratory evidence of DN with a normal genotype were 55.56%, heterozygous 4G/5G were 40.0%, and homozygous 4G/4G were 4.44%. Healthy individuals with a normal genotype were 71.05%, heterozygous 4G/5G – 21.05%, homozygous 4G/4G – 7.9% (chi-square=37.57, p<0.001) (Table 4).

The 24-month tendency of serum creatinine in dependence to 4G/5G polymorphism of the gene for PAI-1 in patients with DM2 and DN was established – in 4G/4G homozygous for PAI-1 serum creatininrison to heterozygous 4G/5G for PAI-1 (118.10–135.74 µmol/l) and those with a normal genotype (101.53–128.86 µmol/l) (p<0.05) (Fig. 4). The analysis showed a very strong correlation between serum creatinine and the 4G/5G polymorphism of the PAI1 – R2 gene is 0.9988 in homozygotes, R2 – 0.9988 in heterozygotes, and R2 – 0.9993 in those with normal genotype (Fig. 4).

We studied the 24-month tendency of albumin/creatinine ratio in morning urine in correlation with polymorphism of the gene for PAI1 in patients with DM2 and DN. In homozygous PAI1 4G/4G albumin/creatinine progressed (0.351–1.514 mg/mmol) much slower in comparison to heterozygous PAI1 4G/5G (0.734–1.384 mg/mmol) and those with a normal genotype (0.727–1.258 mg/mmol). In heterozygous PAI1 4G/5G albumin creatinine ratio (0.734–1.384 mg/mmol) in morning urine progressed much faster in comparison to those with a normal genotype (0.727–1.258 mg/mmol) (p<0.05) (Fig. 5). The analysis showed a very strong correlation between the albumin/creatinine ratio and the 4G/5G polymorphism of the PAI-1 – R2 gene is 0.9988 in homozygotes, R2 – 0.9988 in heterozygotes, and R2 – 0.9988 in those with normal genotype (Fig. 5).

The following results demonstrate the correlation of creatinine clearance and 4G/5G polymorphism of the gene for PAI1 in patients with DM2 and DN at month 6. Creatinine clearance in homozygous 4G/4G for PAI1 4 was the lowest 88.8 ml/min, higher in heterozygous PAI1 4G/5G 99.59 ml/min, and the highest in normal genotype 121 ml/min (p<0.05) (Fig. 6). The analysis showed a very strong correlation between creatinine clearance and the 4G/5G polymorphism of the PAI1 – R2 gene was 0.965 (Fig. 6).

Our study shows a much higher distribution of the genotype 4G/4G for PAI-1 and MTHFR T677T among patients with DN in comparison to groups with DM2 and no DN, compared by sex and age, with a similar duration of the disease and glycemic control. We studied the effect of the 4G/5G genotypes for PAI-1 and С677T for MTHFR on the progression of DN regarding renal function with the use of serum creatinine, creatinine clearance and albumin/creatinine ratio. The genotype PAI-1 4G/5G and MTHFR С677T are independent risk factors for the development of nephropathy in patients with DM2; they have direct correlation with the progression of CKD. 4G/5G polymorphism for PAI-1 and С677T for MTHFR can be used for an early prognostic marker for the development of diabetic nephropathy and CKD in patients with DM2. Our results correspond to the results reported by Chen et al.^[8] summarized in several meta-analyses based on 13 studies with 891 healthy people, 894 with diabetic nephropathy and 1261 with diabetes mellitus without diabetic nephropathy. We found that C677T mutation of MTHFR gene predisposes patients with type 2 diabetes to developing diabetic nephropathy. The T allele of this mutation is related to the more rapid progression of nephropathy to a final stage of kidney failure. Several studies from 2006 and 2020 demonstrated that the 4G/4G polymorphism of the plasminogen activator inhibitor type 1 gene increases the risk for developing diabetic nephropathy in patients with type 2 diabetes mellitus. This polymorphism is related to rapid progression of nephropathy to a final stage of kidney failure, which is fully consistent with our results.^[11–14]

C677T polymorphism of the MTHFR gene is associated with the increase of serum creatinine levels. The presence of a T allele in the MTHFR gene determines the tendencies for increasing serum creatinine, lowering creatinine clearance and increasing albumin/creatinine ratio in morning urine. The 4G/5G polymorphism of PAI1 gene is associated with an increase in serum creatinine. The presence of a 4G allele in the PAI1 gene determines the tendencies for increasing serum creatinine, lowering creatinine clearance, and increasing albumin/creatinine ratio in morning urine.