Objective: Diabetic kidney disease DKD (Diabetic nephropathy DN) is considered one of the chronic micro vascular complications of diabetes mellitus and considered the commonest cause leading to chronic renal failure and chronic renal dialysis. Genetic susceptibility has been implicated in DKD. The angiotensin converting enzyme (ACE) is one of the key roles in the renin angiotensin system cascade by converting angiotensin I to angiotensin II which plays a key role in regulation of blood pressure as well as electrolytes and fluid balance. This study addressed the association of (ACE) gene polymorphisms with DN in Egyptian (T2DM) patients. Methods: Our research comprised of 75 cases of T2DM with diabetic kidney disease, 100 cases of T2DM without DKD and 94 healthy volunteers. Different genotypes of ACE gene were determined by SSP-PCR analysis. Results: Gene polymorphism of ACE (DD, ID, II) in diabetic patient with DKD is 44%, 52%, 4% respectively and for T2DM individuals without DKD is 23%, 72%, 5% respectively. (DD) had significant higher frequencies in T2DM patients with DKD compared to those without DKD (p < 0.005) and (ID) had significant higher frequencies in T2DM without DKD (p < 0.0001). These results indicated that there is an association between ACE gene polymorphisms and susceptibility of diabetic patients to be affected by diabetic kidney disease. Conclusion: From our results, we can conclude that genotype of ACE in Egypt DD is the genotype of cases diabetic kidney disease. So the presence of D allele has a significant relation with diabetic kidney disease. Our data confirm the role of ACE in its relationship with diabetic kidney disease in Egyptian type 2 diabetic patients.
Diabetic kidney disease DKD (Diabetic nephropathy DN) is a clinical syndrome characterized by persistent albuminuria (>300 mg/d or >200 μg/min) that is confirmed on at least 2 occasions 3 - 6 months apart, progressive decline in the glomerular filtration rate (GFR) and elevated arterial blood pressure [
Diabetic kidney disease is considered one of the most common causes for chronic renal failure and chronic hemodialysis [
Gene insertion (I), deletion (D) polymorphism within the human ACE gene [
Angiotensin II (Ang II) considered a very strong vasoconstriction factor of the systemic and the local blood pressure [
Several polymorphisms depend on the presence or absence of a 287 base pair sequence in intron 16, three main different genotypes homozygotes (DD, II) and heterozygote ID are found [
The aim of our research was to check for the association of ACE gene polymorphisms with the susceptibility to Diabetic kidney disease in Egyptian individuals with T2DM.
This research has included 175 subjects with type 2 Diabetes Mellitus. They were recruited from the Internal Medicine Hospital (Diabetes clinic), Mansoura University, Egypt in the period between May and December 2017. The ethics committee approved the study protocol and the study was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all the patients included in the study.
An inclusion criterion includes type 2 Diabetic patients fulfilling criteria of diabetic nephropathy. An exclusion criterion includes, Type 1 diabetes mellitus, gestational diabetes, secondary diabetes, associated autoimmune diseases, non diabetic kidney diseases, patients suffering from hematuria, acute infections particularly urinary tract infections, and pregnant females were excluded from the study.
Selected Diabetic patients divided into 2 groups according to the presence of nephropathy consists of 75 subjects affected with diabetes type 2 associated with DKD compared to 94 healthy volunteers. In the Type 2 diabetic patients affected with Diabetic kidney disease, the mean ± SD age was 58.03 ± 6.34 ranges from 45 to 74 years. They were in the form of 38 (50.7%) males and 37 (49.3%) females. The other group was affected with diabetes type 2 without DKD (n = 100), their mean ± SD age was 52.1900 ± 8.31901 range from 34 to 75 years; the gender divided between 30 (30.0%) males and 70 (70.0%) (
All subjects were questioned about history of diabetes mellitus, hypertension, hypercholesterolemia, history of DKD in the first degree relatives. Regarding clinical examination, blood pressure, weight, heights were measured. Laboratory investigation was done and included detection of urinary albumin with the following cutoff values (microalbuminuria, (Albumin/creatinine ration (ACR) between 30 - 300 mg/g) and macroalbuminuria, (ACR more than 300) (according to national kidney foundation), detection of Glycated hemoglobin (HbA1c), lipid profile (
Groups | N | Mean | |
---|---|---|---|
Age | T2DM with neuropathy | 75 | 58.03 ± 6.34 |
T2DM without neuropathy | 100 | 52.1900 ± 8.31901 | |
Health control | 94 | 51.179 ± 9.217 | |
Sex | Male | Female | |
T2DM with neuropathy | 50.7% | 49.3% | |
T2DM without neuropathy | 30% | 70% | |
Health Control | 46.8% | 53.2% | |
Body mass index (kg/rn2) | T2DM with neuropathy | 75 | 28.3 ± 4.1 |
T2DM without neuropathy | 100 | 28.6 ± 5.2 | |
Health control | 94 | 26.7 ± 3.6 |
Data are means and SD.
Parameters | Groups | Mean ± SD | ||
---|---|---|---|---|
duration of diabetes (Years) | T2DM with neuropathy | 14.85 ± 5.032 | ||
T2DM without neuropathy | 14.74 ± 4.15 | |||
HbAlc (%) | T2DM with neuropathy | 8.4 ± 1.2 | ||
T2DM without neuropathy | 8.1 ± 1.6 | |||
Retinopathy | T2DM with neuropathy | Background | 52/75 (69.33%) | |
Proliferative | 21/75 (28%) | |||
T2DM without neuropathy | Background | 18/100 (18%) | ||
Proliferative | 4/100 (4%) | |||
Creatinine (mg/dl) | T2DM with neuropathy | 1.2 ± 0.9 | ||
T2DM without neuropathy | 0.8 ± 0.2 | |||
Cholesterol (mg/dl) | T2DM with neuropathy | 210.22 ± 69.16 | ||
T2DM without neuropathy | 155 ± 44.32 | |||
Triglyceride (TG, mg/dl) | T2DM with neuropathy | 140.97 ± 88.60 | ||
T2DM without neuropathy | 133 ± 45.23 | |||
Blood Pressure | T2DM with neuropathy | Mild hypertension | 35 (45.9%) | |
Moderate hypertension | 28 (37.9%) | |||
Severe hypertension | 8 (10.8%) | |||
Normal | 4 (5.4%) | |||
T2DM without neuropathy | Mild hypertension | 31 (31%) | ||
Moderate hypertension | 23 (23%) | |||
Severe hypertension | 9 (9%) | |||
Normal | 37 (37%) | |||
High density lipoprotein (HDL, mg/dl) | T2DM with neuropathy | 39.14±12.67 | ||
T2DM without neuropathy | 42.11 ± 6.32 | |||
Low density lipoprotein (LDL, mg/dl) | T2DM with neuropathy | 144.35 ± 63.21 | ||
T2DM without neuropathy | 111.24 ± 24.105 | |||
Microalbuminuria/ Macroalbuminuria | T2DM with neuropathy | 20/42 | ||
T2DM without neuropathy | - | |||
Data are means and SD.
At first taking informed consent from all diabetic individuals included in our research and healthy volunteers, venous blood samples (3 ml) were withdrawn and added on EDTA (ethylenediamine tetra acetate) containing tubes, DNA was extracted promptly using DNA extraction and purification kit (Gentra Systems, USA) according to manufacturer’s instructions and then stored at −20˚C till use.
ACE genotype analysis was performed by PCR-RFLP analysis.
Genomic DNA was isolated from peripheral blood leukocytes according to a standard salting out method [
Data were prepared and undergoing analysis through Statistical Package of Social Science (SPSS, version 10.0). The frequencies of different allelic polymorphisms of all studied individuals were compared between groups by using Fisher’s exact test (modified Chi square test) and Odds ratio. A value of p < 0.05 was considered to be significant.
Comparing studied cases of T2DM with nephropathy to that of T2DM without nephropathy regarding the gene polymorphism of ACE and alleles (
By comparing cases of T2DM without nephropathy versus healthy people (
Comparing cases albuminuria >300 with those <300 as regards the studied ACE gene polymorphisms, it is observed that cases >300 have high frequency of DD genotype (45.2% vs. 30%, OR = 1.93, p = 0.75). Also cases >300 had low frequency of ID genotype (50% vs. 65%, OR = 0.54, p = 0.403). Regarding the allele frequencies, the D allele showed higher level among cases with macroalbuminuria (70.24% vs. 62.5%, OR = 1.42, p = 0.51), while the I allele showed lower level (29.76% vs. 37.5%, OR = 0.71, p = 0.51) (
Comparing cases with blood pressure groups regards that the studied ACE gene polymorphisms, it is observed that in DD genotype normal have high level then sever then mild and moderate is the lower one (75%, 50%, 44.1%, 39.3, p = 0.232). In addition, in ID genotype found that moderate have higher level then mild then sever and normal is lower one (57.1%, 52.9%, 50%, 0%, p = 0.232) (
Comparing cases with hyperlipidemia with those without hyperlipidemia as regards the studied ACE gene polymorphisms, it is observed that cases with hyperlipidemia have high frequency of DD genotype (46.2% vs. 44.7%, OR = 1.06, p = 0.903). In addition, cases with hyperlipidemia had high frequency of ID genotype (53.8% vs. 48.9%, OR = 1.22, p = 0.874). Regarding the allele frequencies, the D allele showed higher level among cases with hyperlipidemia (46.2% vs. 44.7%, OR = 1.06, p = 0.903), while the I allele showed lower level (26.9% vs. 30.9%, OR = 0.83, p = 0.76) (
OR (95% CI) | χ2 (P) | Diabetic subjects without nephropathy n (%) | Diabetic subjects with nephropathy n (%) | |
---|---|---|---|---|
N = 100 | N = 75 | |||
2.6 (1.37 - 5.05) | 7.75 (0.005)* | 23 (23.0) | 33 (44.0) | DD |
0.27 (0.14 - 0.54) | 13.32 (<0.0001)** | 72 (72.0) | 39 (52.0) | ID |
0.792 (0.18 - 3.42) | 0.003 (0.956) | 5 (5.0) | 3 (4.0) | II |
N = 200 | N = 150 | Alleles | ||
1.62 (1.035 - 2.54) | 4.023 (0.045)* | 118 (59) | 105 (70) | D |
0.62 (0.39 - 0.97) | 4.023 (0.045)* | 82 (41) | 45 (30) | I |
Diabetic subjects without nephropathy n (%) | Healthy subjects n (%) | χ2 (P) | OR (95% CI) | |
---|---|---|---|---|
N = 100 | N = 94 | |||
DD | 23 (23.0) | 46 (48.9) | 13.11 (<0.0001)** | 0.31 (0.17 - 0.58) |
ID | 72 (72.0) | 43 (45.7) | 12.77 (<0.0001)** | 3.05 (1.7 - 5.54) |
II | 5 (5.0) | 5 (5.3) | 0.05 (0.823) | 0.94 (0.26 - 3.35) |
Alleles | N = 200 | N = 188 | ||
D | 118 (59) | 135 (71.8) | 6.45 (0.011)* | 0.57 (0.37 - 0.86) |
I | 82 (41) | 53 (28.2) | 6.45 (0.011)* | 1.77 (1.16 - 2.71) |
n = number of cases, (%) = percentage of cases, Odds ratio & 95% confidence interval = OR (95% CI). * p < 0.05 (significant) ** p < 0.001 (extremely significant): Significance using Fisher’s exact test.
T2DM with Nephropathy n = 75 (%) | Healthy Control n = 94 (%) | X² (p) | OR 95% CI | |
---|---|---|---|---|
DD | 33 (44.0) | 46 (48.9) | 0.234 (0.629) | 0.82 (0.45 - 1.51) |
I D | 39 (52.0) | 43 (45.7) | 0.43 (0.51) | 1.29 (0.7 - 2.4) |
I I | 3 (4.0) | 5 (5.3) | 0.001 (0.98) | 0.74 (0.17 - 3.21) |
Allele | N = 150 | N = 188 | ||
D | 105 (70) | 135 (71.8) | 0.059 (0.808) | 0.92 (0.57 - 1.47) |
I | 45 (30) | 53 (28.2) | 0.059 (0.808) | 1.092 (0.68 - 1.75) |
Comparing cases with diabetic nephropathy with those healthy controls as regards the studied ACE gene polymorphisms, it is observed that cases with nephropathy lower frequency of DD genotype, which was not significant from that of healthy controls (44% vs. 48.9%, OR = 0.82, p = 0.629). Also cases of nephropathy had higher frequency of ID genotype (52% vs. 45.7%, OR = 1.29, p = 0.51). Regarding the allele frequencies the D allele showed lower level among cases diabetic nephropathy (70% vs. 71.8%, OR = 0.92, p = 0.808), while the I allele showed higher level (30% vs. 28.2%, OR = 1.09, p = 0.059).
Macroalbuminuria N = 42 (%) | Microalbuminuria N = 20 (%) | X2 (p) | OR 95% CI | |
---|---|---|---|---|
DD | 19 (45.2) | 6 (30.0) | 0.75 (0.39) | 1.93 (0.62 - 5.99) |
I D | 21 (50.0) | 13 (65.0) | 0.7 (0.403) | 0.54 (0.18 - 1.62) |
I I | 2 (4.8) | 1 (5.0) | 0.35 (0.55) | 0.95 (0.081 - 11.14) |
Alleles | N = 84 | N = 40 | ||
D | 59 (70.24) | 25 (62.5) | 0.43 (0.51) | 1.42 (0.64 - 3.13) |
I | 25 (29.76) | 15 (37.5) | 0.43 (0.51) | 0.71 (0.32 - 1.56) |
Comparing cases albuminuria >300 with those <300 as regards the studied ACE gene polymorphisms, it is observed that cases >300 have high frequency of DD genotype (45.2% vs. 30%, OR = 1.93, p = 0.75). Also cases >300 had low frequency of ID genotype (50% vs. 65%, OR = 0.54, p = 0.403). Regarding the allele frequencies, the D allele showed higher level among cases with macroalbuminuria (70.24% vs. 62.5%, OR = 1.42, p = 0.51), while the I allele showed lower level (29.76% vs. 37.5%, OR = 0.71, p = 0.51).
Blood. p | X2 (p) | ||||
---|---|---|---|---|---|
Mild N = 34 (%) | Moderate N = 28 (%) | Severe N = 8 (%) | Normal n = 4 (%) | ||
DD | 15 (44.1) | 11 (39.3) | 4 (50.0) | 3 (75.0) | 8.090 (0.232) |
I D | 18 (52.9) | 16 (57.1) | 4 (50.0) | 0 (0) | |
I I | 1 (2.9) | 1 (3.6) | 0 (0) | 1 (25.0) |
Comparing cases with blood pressure groups regards that the studied ACE gene polymorphisms, it is observed that in DD genotype normal have high level then sever then mild and moderate is the lower one (75%, 50%, 44.1%, 39.3, p = 0.232). In addition, in ID genotype found that moderate have higher level then mild then sever and normal is lower one (57.1%, 52.9%, 50%, 0%, p = 0.232).
With Hyperlipidemia N = 26 (%) | Without Hyperlipidemia N = 47 (%) | X2 (p) | OR 95% CI | |
---|---|---|---|---|
DD | 12 (46.2) | 21 (44.7) | 0.015 (0.903) | 1.06 (0.41 - 2.78) |
I D | 14 (53.8) | 23 (48.9) | 0.025 (0.874) | 1.22 (0.47 - 3.18) |
I I | 0 (0) | 3 (6.4) | 0.22 (0.64) | 0.24 (0.012 - 4.83) |
Allele | N = 52 | N = 94 | ||
D | 38 (73.1) | 65 (69.1) | 0.096 (0.76) | 1.21 (0.57 - 2.57) |
I | 14 (26.9) | 29 (30.9) | 0.096 (0.76) | 0.83 (0.39 - 1.75) |
Comparing cases with hyperlipidemia with those without hyperlipidemia as regards the studied ACE gene polymorphisms, it is observed that cases with hyperlipidemia have high frequency of DD genotype (46.2% vs. 44.7%, OR = 1.06, p = 0.903). In addition, cases with hyperlipidemia had high frequency of ID genotype (53.8% vs. 48.9%, OR = 1.22, p = 0.874). Regarding the allele frequencies, the D allele showed higher level among cases with hyperlipidemia (46.2% vs. 44.7%, OR = 1.06, p = 0.903), while the I allele showed lower level (26.9% vs. 30.9%, OR = 0.83, p = 0.76).
There are several researches indicated that development and progression of diabetic kidney disease are multifactorial including different pathophysiologic mechanisms especially environmental or genetic susceptibility. Epidemiological studies found familial clustering of diabetic kidney disease in diabetic siblings, supporting an important role of genetic defects in the pathogenesis of diabetic kidney disease [
This study included (269) 75 patients with diabetic kidney disease (DKD), 100 patients T2DM patients without DKD and 94 healthy people. In selection of cases, we were keeping to have cases affected with T2DM associated with diabetic kidney diseases. Their mean age was 58.02 years, with a SD of ±6.34 years. Out of them, 94.6% having hypertension, and 35.6% with hyperlipidemia and 22.5% have consanguinity and 72% have family history to diabetes. For comparison 100 cases diabetic (T2DM) without nephropathy (mean age was 51.7 years, with a SD of ±9.4 years) (
This study showed that Egyptian cases of diabetic kidney disease had significantly higher frequency of genotype (DD) than cases diabetic with no DKD (44% versus 23%, p = 0.005). In addition, it’s noticed that cases of diabetic nephropathy had significantly lower frequency of ID genotype than cases of diabetes without nephropathy (52% versus 72%, p < 0.0001) (
Diabetic nephropathy cases showed low frequency of II genotype than diabetic without nephropathy 5.3% (4% vs. 5%). Meanwhile, total cases found to have statistically significant more frequent expression of D allele (70% vs. 59%, p = 0.045) with a significant lower level of I allele than cases of diabetic without nephropathy (30% vs. 41%, p = 0.045). Finally, in both group of cases and controls we observed that frequency D allele is higher than frequency of I allele.
In a previous study among Egyptian cases, reported that the II, ID and DD ACE genotypes was 4%, 52% and 44% in cases of diabetic nephropathy and 5%, 72% and 23% in cases without nephropathy. The ACE DD genotype shows significant association with diabetic nephropathy. We can speculate that the difference may be related to the ethnic background variations between our locations in Egypt and other countries.
Analyzing the results among Egyptian controls, this study showed that Egypt control cases (diabetic without nephropathy) had a higher frequency of ID than that of DD genotype (72% vs. 52%) with a higher frequency of II genotype (5% vs. 4%).
Also, our results showed that by comparing cases albuminuria >300 with those <300 as regards the studied ACE gene polymorphisms, it is observed that cases >300 have high frequency of DD genotype (45.2% vs. 30%, OR = 1.93, p = 0.75). Also cases >300 had low frequency of ID genotype (50% vs. 65%, OR = 0.54, p = 0.403).
Regarding the allele frequencies, the D allele showed higher level among cases with macroalbuminuria (70.24% vs. 62.5%, OR = 1.42, p = 0.51), while the I allele showed lower level (29.76% vs. 37.5%, OR = 0.71, p = 0.51) (
These findings are supported by study carried out in India population by [
Regarding other micro vascular complications related to diabetic kidney disease [
Comparing cases with blood pressure groups regards that the studied ACE gene polymorphisms, it is observed that in DD genotype normal have high level then sever then mild and moderate is the lower one (75%, 50%, 44.1%, 39.3%, p = 0.232). In addition, in ID genotype found that moderate have higher level then mild then sever and normal is lower one (57.1%, 52.9%, 50%, 0%, p = 0.232) (
In South Korea, subjects with genotype DD compared to others with genotype II, the OR was 3.881 (95% confidence interval, 1.564 9.628; p = 0.003 approximately), these results indicated that the DD genotyping of ACE gene may be considered a significant risk factor for the progressive nature of diabetic kidney disease.
Also, [
Also, in Tokyo, [
Also these findings were in contrast with [
In addition, [
In addition, [
In addition, In Poland, [
In addition, In Germany, [
Comparing cases with hyperlipidemia with those without hyperlipidemia as regards the studied ACE gene polymorphisms, it is observed that cases with hyperlipidemia have high frequency of DD genotype (46.2% vs. 44.7%, OR = 1.06, p = 0.903). In addition, cases with hyperlipidemia had high frequency of ID genotype (53.8% vs. 48.9%, OR = 1.22, p = 0.874).
Regarding the allele frequencies, the D allele showed higher level among cases with hyperlipidemia (46.2% vs. 44.7%, OR= 1.06, p = 0.903), while the I allele showed lower level (26.9% vs. 30.9%, OR= 0.83, p = 0.76) (
Possible explanation of this controversy in the results of different researches related to genetic polymorphisms may be due to multifactorial aspects, mainly the major differences in ethnic aspects of studied diabetic individuals and healthy volunteers. Other factors include the definition of nephropathy or Diabetic kidney disease and inclusion criteria of the diabetic control group without renal complication and small sample sizes in some studies.
Our findings indicated that there is a strong relation between diabetic kidney disease and genetic polymorphism of ACE gene and from our results we also found that genotype of ACE in Egypt is DD genotype of diabetic cases with diabetic kidney disease so the presence of D allele has a significant relation with diabetic kidney disease. Our data confirm the significant role of angiotensin converting enzyme gene in its relationship with diabetic kidney disease risk in Egyptian population.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. All authors sharing in this manuscript have no conflicts of interest. All authors have contributed significantly, and that all authors are in agreement with the content of the manuscript.
The authors declare no conflicts of interest regarding the publication of this paper.
El-baz, R.A., Wafa, A.M., Marrawan, El-Sh., El-Tawab, A.R.A., and Aly, Z.I. (2018) Study of Angiotensin Converting Enzyme Gene Polymorphism in Egyptian Type 2 Diabetes Mellitus with Diabetic Kidney Disease. International Journal of Clinical Medicine, 9, 629-643. https://doi.org/10.4236/ijcm.2018.98053
T2DM: type 2 diabetes mellitus
DN: diabetic nephropathy
ACE: Angiotensin-converting enzyme
SSP-PCR: sequence specific primer-polymerase chain reaction