Association of renin-angiotensin system genes polymorphism with progression of diabetic nephropathy in patients with type 1 diabetes mellitus

Background/Aim. Diabetic nephropathy (DN) as a major microvascular complication of diabetes mellitus (DM) include a progressive increase in urinary albumin excretion in association with an increase in blood pressure and to end stage renal failure. Hypertension connected with renin-angiotensin system (RAS) hyperactivity and corresponding genotypes, angiotensinogen (AGT), angiotensine-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1R), predispose the increasing risk of DN. The aim of this study was to assess the distribution of AGT, ACE and AT1R gene polymorphisms in patients with type 1 DM according to the level of DN and patients clinical characteristics. Methods. The study included 79 type 1 diabetic patients. Inclusion criteria were: age between 20–40, duration of diabetes > 5 years, and no other severe diseases. Clinical characteristics were gained from interviewing the patients. Polymorphism was detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism using restriction enzymes Psy I (Tth 111 I) and Hae III. Results. The patients with proteinuria compared with normoand microalbuminuric patients, highly differed in age, diabetes duration, blood pressure level, hypertension, rethynopathy and urinary albumin excretion values (p < 0.001). No statistically significant difference between the groups was found for the ACE and AT1R gene polymorphisms distribution. The presence of TT genotype of the M235T polymorphism was significantly higher in the group with proteinuria (p < 0.05). The patients with hypertension raised nephropathy 5.2 times higher (OR = 5.20, p < 0.05) while carriers of TT allel developed nephropathy 28.38 times higher (OR = 28.389, p < 0.01) than those with MM genotype. Conclusion. Increased association of hypertension and TT angiotensinogen gene polymorphism in patients with diabetes mellitus with proteinuria could be a significant marker of diabetic nephropathy.

DN is defined as increased protein excretion in urine.Early stage is characterized by a small increase in urinary albumin excretion (UAE), also called microalbuminuria or incipient DN.More advanced disease is defined by the presence of macroalbuminuria or proteinuria.The latter is named overt or "manifested" DN.
Patients with elevated level of urinary albumin excretion are at high risk of cardiovascular complications and need frequent and carefull examination for early detection of nephropathy and cardiovascular and lipid abnormalities 2 .
Hemodynamic changes in DN, as systemic and glomerular hypertension, lead to the initiation and progression of nephropathy and may be explained by alteration in the renin-angiotensine system (RAS).
The prominent role of the component genes of the RAS in cardiovascular regulation sugest the possibility that polymorphism of the genes of the RAS might be involved in the genetic predisposition to develop DN.Among the candidate genes of the RAS, the angiotensinogen (AGT), ACE and angiotensin II type 1 receptor (AT1R) genes seem to be particulary relevant to renal disease and related with predisposition for renal complications 3,4 .The genetic polymorphisms of these key components of RAS provide a basis for studying the relationship between genetic variants and the development of vascular and renal damage in patients with DM.
Several polymorphisms were identified in the AGT gene wich are linked to essential hypertension.Of those, M235T polymorphism (methionine substituted by threonine) resulting in M and T alel forms, was extensively studied in cardiovascular and renal diseases 4,5 .A TT genotype is associated with the progression of limited hypertension into manifested hypertension as well as with increasing risk for the diabetic nephropathy level 6 .
Polymorphism of the ACE gene has a frequent insertion-deletion (I/D) polymorphism characterized by the 278bp insertion (allele I) or deletion (allele D) variant in intron 16 associated with serum and tissue ACE levels.The I/D polymorphism, wich determine most of ACE individual variance was proposed as a genetic marker for DN.The level of ACE in plasma is in direct correlation with the genotype, and plays a critical role in determining intrarenal angiotensin and kinin concentrations 7 .
The AT1R polymorphism located at the position 1166 (A/C) in 3 untranslated region has been considered as a risk factor for hypertension and cardiovascular disease 5 .
In the present study we examined the distribution of the AGT, ACE and AT1R gene polymorphisms in patients with type I DM devided into three groups according to the level of DN -normoalbuminuric, microalbuminuric or incipient nephropathy and proteinuric or macroangiopathy level.Also, polymorphisms of three studied genes were correlated with clinical characteristics of patients with DN.
This study was approved by the Ethic Committee of the Institute of Diabetes and Metabolic Diseases, Clinical Center of Serbia, Belgrade.

Methods
All the patients with type 1 DM were presented with certain criteria and hospitalised in the Institute of Diabetes and Metabolic Diseases from 2008 to 2010.
The prospective observational study included 79 type 1 diabetic patients, among them 33 had normoalbuminuria, 21 were with microalbuminuria, while 25 patients had proteinuria.The patients were classified according to the amount of albuminuria using the median result of the three 24 h urine collections.Urinary albumin excretion (UAE) was determined with the nephelometric technique.Normoalbuminuric level was defined as UAE < 30 mg/24 h, microalbuminuria as UAE 30-300 mg/24 h and macroalbuminuria or manifested proteinuria as UAE over 300 mg/24 h.Inclusion criteria were age (20-40 years), duration of diabetes (> 5 years) and the absence of other severe diseases.
Clinical characteristics were gathered from interviewing the patients (duration of diabetes, family history for hypertension, cardiovascular diseases, diabetes, smoking duration).The defined risk factors (arterial blood pressure and complications) were measured on the first day of visit.Blood samples were drawn for measurement of serum cholesterol, HbA1c and for the determination of genotypes.Strana 629 Ili V, et al.Vojnosanit Pregl 2014; 71 (7): 627-633.

Determination of genotypes
Individual genomic DNA samples were extracted from periferal blood with Applied Biosystems 6100 Nucleic Acid prep Station instrument.
The AGT (M235T) gene polymorphism and AT1R (A1166C) gene polymorphisms were analysed by polymerase chain reaction (PCR) and subsequent restrictionendonuclease digestion (Tth 111I and Hae III) 8 .To determine the ACE I/D genotype, DNA (100 ng) was amplified using two insertion-specific primers described by Bonnardeaux et al. 9 .The reaction products were visuelised on 2% agarose gel stained with ethidium bromide.Final analysis of the genotype was performed after 10% PAGE stained with silver nitrate.

Statistical analysis
All statistical analyses were performed in SPSS 12.0 (SPSS Inc, Chicago, Illinois) statistical package).The results are presented as frequency, percent and mean ± SD.The 2 , Kruskal-Wallis and ANOVA test were used to compare the 3 groups, while Mann-Whitney U-test was used to compare the 2 groups.A logistic regression model was performed to asses associations of nephropathy and other variables.All p-values that were less than 0.05 were considered significant.

Results
Patient characteristics (total number 79) are given in Table 1.
The patients with proteinuria, compared with normoand microalbuminuric patients, highly differed in age and diabetes duration, blood pressure level and hypertension.
There was no statistically significant diference in the occurence of polyneuropathy among the examined groups of patients, while the presence of rethinopathy as a frequent mi-crovascular complication showed high statistically significant difference in the group of patients with proteinuria (Table 1).
We genotyped all the patients for the AGT, ACE and AT1R gene polymorphisms.The distribution of genotype and allele frequencies in the examined groups are presented in Table 2.
No statistically significant difference among the examined groups was found in the genotype and allele distribution for the ACE and AT1R gene polymorphisms.
The carriers of the MT and TT genotype of the AGT gene polymorphism were more frequent in the group of patients with elevated UAE.The distribution of homo-and heterozygous genotypes for the T allele (MT and TT) was significantly higher in the group of patients with proteinuria when compared to normoalbuminuric patients.
The presence of homozygous TT genotype of the AGT M235T polymorphism, compared among the 3 devided groups according to UAE level, was significantly higher in the group of patients with high albumin excretion.
The genotype and allele distribution for the ACE and AT1R gene polymorphisms, among the examined groups according to elevated albumin excretion, showed no statistically significant difference.
We found the tendency for statistical significance between the pressence of TT and I/D gene polymorphisms and increased blood pressure in patients with proteinuria.The pressence of MT/TT genotype of AGT M235T gene and AC/CC genotype of AT1R gene did not reveal a significant association with retinopathy in the group of patients with proteinuria, but the homozygous DD genotype of the ACE gene polymorphism showed an increasing frequency in the proteinuric group compared with normoalbuminuric patients (Table 3).
Using the Backward logistic regression model, after obtaining all predictors associated with renal failure, we   found that duration of diabetes, high blood pressure, rethinopathy and AGT polymorphism were highly associated with nephropathy in patients with DM (Table 4).
It was shown that as the duration of diabetes increases by one unit, the chances of developing nephropathy were 1.192 times higher being statistically significant.If a patient had hypertension, the likelihood for nephropathy was 5.2 times higher.A patient with TT allele proned to develop nephropathy 28.38 times higher than the carrier of MM genotype.If a patient had the MT allele, the chance to develop nephropathy was 1.358 times greater, but this was not statistically significant.In fact, only the TT genotype showed notable importance.
The univariate analyses did not show a statistically significant association for ACE/II genotype according to increasing renal complications.The DD genotype in our study is associated with 0.307 lower risk for nephropathy.

Discussion
We examined the association between the DN level and genotype distribution of the RAS genes in normoalbuminuric, micro and proteinuric groups of patients with DM type 1.Also, we analysed main clinical complications in patients with DM type 1 and correlated them with the studied gene polymorphisms.
Patients with microalbuminuria are at high risk of cardiovascular complications and need frequent and careful examination for early detection of nephropathy, retinopathy, cardiovascular and lipid abnormalities.Identification of factors related to the development of microalbuminuria leads to the development of strategies to prevent nephropathy and reduce the occurence of new cases 8 .
Our data suggest a statisticaly significant correlation between blood pressure level (systolic and diastolic) and rethinopathy as more prominent microvascular complication in patients with terminal renal insuficiency.Both systolic and diastolic hypertension accelerate the progression of diabetic nephropathy, and agressive antihypertensive management successed to decrease the rate of fall of glomerular filtration rate (GFR) 10 .
In patients with type 1 diabetes, the median life expectancy can significantly increase using appropriate antihypertensive intervention, with the reduction of mortality from 94% to 45%, and the reduction in the need for dialysis and transplantation from 73 to 31% 16 years after the development of overt nephropathy 11 .For patients with nephropathy, treatment with ACE inhibitors is indicated as a part of initial therapy concerning previous genotyping analysis of the ACE gene polymorphism.
In the present study, due to a rather small number of patients, T allele of the M235T polymorphism and D allel of the ACE I/D polymorphism tend to be increased in patients with elevated blood pressure in the proteinuric type 1 diabetes group.Furthermore, current literature suggests that the ACE D/D genotype predicts poor renal response to ACE inhibitors and to agents that do not block the RAS in subjects with DN.Two longitudinal observational studies pointed out that GFR decreased rapidly in proteinuric type 1 DM patients with the DD genotype 12,13 .In the other study, the albumin excretion rate of type 1 DM patients with the II genotype increased more rapidly than in those with the DD genotype, while the response to ACE inhibitor was better in patients with II than in those with the DD genotype 14 .
In some follow-up studies, implementation of RAS blockade therapy in type 1 diabetic patients reduced the progression to overt DN to 3.4% per year.It should be notified that 45% of patients in progression of DN decrease microalbuminuria by intensified antihypertensive treatment 15 .
Several data suggest that hyperglicaemia may be very important factor for the increasing incidence of cardiovascular complications and progression from microalbuminuric to DN.Despite previous observational studies findings about association of poor glycaemic control and progression in albuminuria and the rate of progression to DN, we found no statistically significant correlations in HbA1c values and quality of glycoregulation between the normoalbuminuric and microalbuminuric patients.There was no significant increasing serum levels of total cholesterol, low density hipoprotein (LDL), high density lipoprotein (HDL) cholesterol in our examined group although it has been reported that dia- betic patients could manifest alterations in serum lipid level ranging from 30 to 90% 16 .Polymorphisms for different components of RAS have been described with controversal results according to the different ethnic backgrounds of the study populations, for example Asian carriers of 235T allel appear to have a higher risk of hypertension than white carriers 17 .
On the basis of the previous studies, we cannot conclude that there is a link between the ACE I/D polymorphism and DN.The results differ in a number of studies and data do not exclude a pathogenic role of mutations somewhere else in the ACE gene, but prevaling conclusion that the ACE I/D polymorphism is not a reliable marker for the prediction of DN in patients with type 1 diabetes 18 .Our data do not indicate an independent effect of ACE and AT1R polymorphism on the risk for DN, so we are unable to confer the interaction between the ACE I/D and M235T polymorphism, suggesting that geneticaly determined AGT levels can cause risk for DN through Ang I generation.
Previous results in type 1 diabetic patients showed an association between DN and the T allele of AGT M235T polymorphism.In the article of Walder et al. 18 , the autors therefore found that the TT genotype was 3 times more common in the group of 98 nephropathic than in 98 normoalbuminuric type 1 DM patients, indicating a link to this genotype and elevated serum angiotensinogen with the possible risk for DN conferred by this genotype.In the another study group of hypertensive patients at a start of dialysis, the carriers of the T allele of the AGT gene were more frequent in the group of patients compared to controls 6,19 .The results of our study showed that in patients with hypertension, a likelihood for nephropathy is 5.2 times higher, while in the group of patients with TT allele risk to develop nephropathy is 23.38 times higher than in the carriers of MM genotype.
Our data designate that the T allele occurs more frequently among patients with proteinuria compared with those with normoalbuminuria, and the level of evidence shows statistically highly significant difference.
In our study, the T allele of the AGT-M235T polymorphism and CC genotype of the AT1-A1166C polymorphism was not associated with retinopathy.
A recent meta-analysis, including two studies with type 1 diabetic patients, did not find corellation between the AGT M235T gene polymorphism and diabetic retinopathy.Nevertheless, the associations between RAS activity and retinopathy have been previously reported [20][21][22][23] .
Local changes as a result of the presence of different polimorphisms of RAS are manifested as changes in renal hemodynamics, increased intraglomerular pressure and glomerular filtration rate, as the major determinants of renal function.The interactions between the alleles of the RAS probably play a major role in determining the development of DN.Experimental data might allow identification of groups with high risk of developing diabetic nephropathy and, therefore, the analysis of RAS system genes plays an important role in providing novel therapeutic targets or individualized treatment strategies for both the prevention and treatment of these complications.

Conclusion
The increased association of hypertension and the TT AGT gene polymorphism in patients with diabetes mellitus with proteinuria could be a significant marker of diabetic nephropathy.

Table 2 Distribution of genotypes and allele frequencies of AGT, ACE nad AT1R genes in the examined patients
a Mann-Whitney U test; b Kruskal-Wallis test; c 2 test; AGT -angiotensinogen; ACE -angiotensin-converting enzyme; AT1R -angiotensin II type I receptor; n -number of patients in examined groups according to albuminuria (percentages are shown in parentheses); ns -not significant.