Abstract
Homocysteine concentration affected by the activities of the enzymes methylene tetra-hyrdofolate reductase (MTHFR). Polymorphisms in MTHFR gene associated with an impairment of MTHFR activity. Hyperhomocysteinemia is a result of single nucleotide polymorphisms (SNPs) in MTHFR 677 C>T that can cause homocysteine levels in the blood to increase. The purpose of this study is to investigate the relationships between MTHFR C677T (rs1801133) gene polymorphism, changes in homocysteine concentrations and progress of renal impairment in young adult hypertensive patients. Two hundred young hypertensive patients (age 21–24 years) were involved in this study; they were classified into patients with and without renal impairment in addition to 200 age and sex matched healthy controls. All participants were submitted to laboratory investigations as assay of MTHFR gene polymorphism C677T (rs1801133) by PCR/RFLP, determination of lipid profile, homocysteine and folic acid concentrations in addition to urinary albumin creatinine ratio (UACR). The levels of both homocysteine and UACR in the TT genotype patients were higher than those in the CC genotype group. Individuals who carry the T allele were more risky to hypertension and progress to early renal impairment in young age compared with those carrying the C allele [OR 2.02 (1.33–3.08), P < 0.001]. Genetic variants of C677T MTHFR gene and hyperhomocysteinemia may be responsible for rapid progress of renal impairment in Egyptian young age hypertensive patients. TT genotype or T allele may be considered as a predisposing factor for both elevated Hcy levels and the development of renal impairment. This study believed that lowering of homocysteine level can reduce renal impairment of hypertensive patients.
Keywords: MTHFR gene polymorphism, Hypertension, Nephropathy
Introduction
Hypertension is a common chronic health problem of unclear etiology. It is a modifiable risk factor for cardiovascular disease and one of the leading causes of end stage renal disease (ESRD). Therefore early intervention to prevent renal injury in hypertensive population is of immense significance to prevent progression to chronic kidney disease (CKD) and ESRD [1]. Hyperhomocysteinemia (HHcy) has been regarded as risk factor related to hypertension [2] and has emerged as an independent risk factor for the progression of CKD [3]. Experimental studies suggest that HHcy induces glomerular injury through redox signaling pathways and DNA hypomethylation mechanisms. So the control of hyperhomocysteinemia could be the corner stone for controlling hypertension and preventing CKD [4]. Homocysteine (Hcy) is a sulfur-containing amino acid that is generated from an essential amino acid; methionine. Hcy concentration is maintained dynamically by either a trans-sulfuration or re-methylation pathway. The enzyme methionine synthase, with vitamin B12 as a cofactor, re-methylates Hcy back to methionine; folate is utilized in homocysteine remethylation to methionine. Hcy level can also be affected by the activities of the enzymes methylene tetra-hyrdofolate reductase (MTHFR) and cystathione beta-synthase (CBS). CBS controls the trans-sulfuration of Hcy to cystathione, and is dependent on vitamin B6 as a cofactor. The metabolic regulation of Hcy is based on the distribution of available Hcy between re-methylation and trans-sulfuration to cystathionine [5]. Mild to moderate Hyperhomocysteinemia is known to be due to genetic factors like mutation in methylene tetrahydrofolatereductase (MTHFR) genes or due to environmental factors like deficiency of vitamin B12 or folic acid. The presence of the C677T mutation is known to correlate with increased MTHFR thermolability and homozygotes for the C677T allele are predisposed to Hyperhomocysteinemia in context to suboptimal folate status [6]. The MTHFR gene is located on short arm of chromosome 1 (1p36.3), it contains 11 exons ranging in size from 102 to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb (Online Mendelian Inheritance in Man—OMIM accession number: 607093). MTHFR gene polymorphisms located on the 677th amino acid residue in exon 4 of gene producing a mutation from C to T and resulting in the C677T mutation can cause the replacement of alanine by valine at position 222 of the encoded MTHFR protein, thus producing the labile change of MTHFR enzyme lead to impairment of MTHFR activity [7, 8]. Aim of this study to investigate the relationships between polymorphism of MTHFR C677T (rs1801133) gen, change in homocysteine concentration and progress of renal impairment in young adult hypertensive patients.
Subjects and Methods
This is a case–control study that carried out among young adult patients with essential hypertension. The study was approved by the local institutional ethics committee and conformed to the Helsinki declaration, and had been conducted in the departments of Internal Medicine and Clinical Pathology. Four hundred Egyptian young age adults (age 21–24 years) were enrolled in this study. Two hundred of them were diagnosed with essential hypertensive disease (the duration of hypertension was calculated from the first diagnosis of hypertension) they were classified to patients with and without renal impairment according to urinary albumin creatinine ratio (UACR), in addition to 200 of normotensive healthy individuals (blood pressure ≤ 120 mmHg for systole and ≤ 80 mmHg for diastole and no history of hypertension) age and sex matched with patients used as control group.
Inclusion Criteria
Essential hypertension was diagnosed as systolic blood pressure ≥ 140 mm Hg and or diastolic blood pressure ≥ 90 mm Hg. Microalbuminuria was used as early indicator for renal impairment. Albumin/creatinine ratio was used for subgrouping of the hypertension group into (hypertension without renal impairment where albumin creatinine ratio < 30 mg/g and hypertension with nephropathy where albumin/creatinine ratio between 30 and 300 mg/g).
Exclusion Criteria
Patients with secondary hypertension, renal failure, diabetes, B12 and folic acid deficiency or other systemic disease were excluded from the study.
Clinical Assessment
Complete history talking, full clinical examination, Electrocardiogram and renal Doppler for detection of renal stenosis to exclude secondary hypertension to verify the inclusion and exclusion criteria of studied subjects.
Laboratory Investigations Including Routine Investigations
Blood glucose level, renal function tests and lipid profile were measured by Cobas 8000 (Roche diagnostics), determination of urinary albumin excretion (UAE) and urinary creatinine for measurement of urinary albumin creatinine ratio (UACR), and urinary albumin were determined by immunoturbidimetric assay, glycated hemoglobin (HbA1c), serum homocysteine and folic acid were determined on Cobas 6000 series e601 module (Ro diagnostics, German).
Molecular Analysis
Assay of MTHFR C677T (rs1801133) genotypes measurement was done by digestion using restriction enzyme (HinfI) of DNA amplified by the polymerase chain reaction (PCR–RFLP) for all subjects at the start of the study.
Genomic DNA extraction: Genomic DNA was extracted from EDTA whole blood sample using a spin column method according to the protocol (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany). DNA was stored at − 20 °C till the time of use.
PCR amplification : MTHFR C677T was amplified using PCR primer sequences were 5′-CAA-AGG-CCA-CCC-CGA-AGC-3′ for sense primers and 5′-AGG-ACG-GTG-CGG-TGA-GAG-TG-3′ for antisense primers.)Metabion International AG) and puReTaq Ready-to-Go PCR Beads (Amersham Biosciences). Each PCR bead is designed for use in a 25 µl reaction volume (one PCR bead/tube). In each bead 0.5 µl of each primer, 3 µl of template DNA and 21 µl of sterile high quality water were added. The amplification was started using thermal cycler (Gene Amp, PCR system 9700) as follow: Initial precycling denaturation by holding at 94 °C for 5 min, then 32 cycles of denaturation at 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min and a final extension period at 72 °C for 9 min. Amplification yield 198 bp product.
Restriction digests reaction: fragment length polymorphism (RFLP) was detected after digestion of amplified DNA with restriction enzyme endonuclease Hinf1 at 37 °C yielding, uncut fragment of 198 bp for wild CC genotype, two fragments of 175 bp, 23 bp for mutant homozygous TT genotype and three fragments of 198 bp and 175 bp, 23 bp for the heterozygous CT genotype. The digested products were separated by electrophoresis on an ethidium bromide stained 3% agarose gel.
Statistical Analysis
Data were presented as mean ± SD or median and range for continuous variables, frequency for categorical ones. Comparisons between quantitative variables were done using ANOVA or Kruskal–Wallis or Mann–Whitney U test when appropriate. For comparing categorical data Chi squared test was performed. Statistical analysis was carried out with SPSS® statistical software version 16.0 (SPSS Inc., Chicago, IL, USA). p value < 0.05 was considered as significantly different.
Results
Compared to healthy controls, both groups of hypertensive patients (with and without renal impairment) had higher concentrations of homocysteine, triglyceride, total cholesterol, low density cholesterol and blood pressure, moreover, HDL-c levels were significantly lower in these groups compared to control subjects. While, serum creatinine and urea levels, GFR and urinary albumin creatinine ratio were significantly increased only in hypertensive group with renal impairment compared to control group. There were a significant increase of homocysteine, triglyceride, total cholesterol, low density cholesterol, creatinine and urea levels, urinary creatinine clearance and urine albumin creatinine ratio (UACR) in hypertensive patients with nephropathy when compared with the group of patients without nephropathy. Furthermore, levels of HDL-c were significantly decreased in HTN patients with renal impairment as compared to the group without impairment while there was no significance change in folate concentration among all studied groups (Table 1). Genotype distribution of this polymorphism conformed to Hardy–Weinberg equilibrium in the control group and hypertension group, which mean the distribution frequencies, reached genetic equilibrium. The genotypes distributions of the MTHFR gene C677T polymorphism were significantly different in hypertensive patients compared to controls. After age and sex were adjusted, comparison of genotypes frequency, the odds ratio of the TT genotype was 1.84 (95% CI 1.01–3.34, P = 0.04) whereas the odds ratio (OR) of CT genotype was 1.42 (95% CI 0.92–2.19, P = 0.11) by using the CC genotype as wild type so that persons who carried the TT or CT genotype had a higher risk of developing hypertension than did those who carried the CC genotype. The allelic frequency of T allele was significantly higher in the hypertensive patients than controls (P = 0.02). Interestingly, subjects carriers T allele were significantly more likely to develop hypertension 1.45 times compared with non-carriers (95% CI 1.07–1.95, P = 0.02) (Table 2). The genotypes distributions of the MTHFR (C677T) gene polymorphism were significantly different in hypertensive patients with renal impairment compared to hypertensive patient without impairment. Comparison of genotypes frequency, the OR of the TT genotype was 2.35 (95% CI 1.13–5.71, P = 0.024) by using the CC genotype as wild type. While the odds ratio (OR) of CT genotype was 2.16 (95% CI 1.16–4.02, P = 0.015) by using the CC genotype as wild type so that hypertensive patient who carried the TT genotype or CT genotype had a higher risk of developing renal impairment more than patients whose carried CC genotype. Hypertensive patients carrying minor T allele were significantly more likely to develop renal impairment compared with those carrying the C allele the odds ratio (OR) was 2.02 (95% CI 1.33–3.08, P < 0.001) (Table 3). In multiple regression analysis, using urine albumin creatinine ratio (UACR) as a dependent variable and included the prediction of homocysteine, lipid profile, urea, creatinine, and GFR that were associated with risk of renal impairment, results showed that the UACR was positively correlated with serum homocysteine, creatinine, triglyceride and TT genotype while was negatively correlated with glomerular filtration rate. The other indicators were not correlated with the UACR (P > 0.05) (Table 4). Individuals carrying TT genotype had higher levels of total cholesterol, triglyceride, LDL cholesterol, homocysteine, impairment of renal function and increased in blood pressure compared to individuals carrying the CC genotype means while lower creatinine clearance and HDL cholesterol were observed in patients carrying TT genotype compared to CC genotype carrier. Also increased in urinary albumin creatinine ratio was observed in subjects carrying the TT genotype compared with subjects carrying the CC genotype (Table 5).
Table 1.
Clinical and biochemical data of study subjects
| Parameters | Groups | |||
|---|---|---|---|---|
| Control (n = 200) | Hypertensive (HTN) patients | P | ||
| Without renal impairment (n = 100) | with renal impairment (n = 100) | |||
| Age (years) Mean ± SD | 22.3 ± 2.79 | 22.6 ± 1.4 | 23.2 ± 3.8 | > 0.05 |
| Male (%)/female (%) | 96/104 | 52/48 | 48/52 | > 0.05 |
| Duration of hypertension month) | – | 62 ± 7 | 65 ± 3 | > 0.05 |
| homocysteine (µmol/l) | 8.61 ± 1.6a* | 13.49 ± 1.3c* | 19.94 ± 1.9b* | < 0.01 |
| folate (nmol/l) | 24.31 ± 4.02 | 25.76 ± 3.7 | 25.60 ± 2.7 | > 0.05 |
| TG (mg/dl) | 94.3 ± 7.67a* | 164.9 ± 70.52c* | 225 ± 48.57b** | < 0.001 |
| TC (mg/dl) | 168 ± 12.85a* | 201 ± 38.27c* | 254 ± 45.65b** | < 0.01 |
| HDLc (mg/dl) | 58.9 ± 6.69a* | 45.3 ± 10c* | 37.3 ± 6.34b* | < 0.01 |
| LDLc (mg/dl) | 88.4 ± 14.36a* | 123 ± 35.48c* | 172 ± 43.39b** | < 0.01 |
| Urea (mg/dl) | 24.4 ± 4.23a* | 31.6 ± 9.54c* | 52.8 ± 20.4b* | < 0.01 |
| Creatinine (mg/dl) | 0.8 ± .13 | 0.7 ± 0.1c* | 1.7 ± 0.78b* | < 0.01 |
| GFR (ml/min) | 102 ± 7.49 | 100 ± 5.16c* | 71.4 ± 23.66b* | < 0.01 |
| UACR (mg/g) | 7.37 ± 2.06 | 11.67 ± 3.04c** | 209.6 ± 51b** | < 0.001 |
| FBG (mg/dl) | 95 ± 2.26 | 94 ± 4.3 | 96 ± 3.7 | > 0.05 |
| HbA1c | 3.8 ± 0.26 | 4.1 ± 0.15 | 4.3 ± 0.17 | > 0.05 |
| SBP (mmHg) | 108 ± 7.2a* | 154.6 ± 9 | 157.3 ± 6.6b* | < 0.01 |
| DBP (mmHg) | 74.6 ± 4.2a* | 94 ± 5.2 | 95.6 ± 4.3b* | < 0.01 |
DBP diastolic blood pressure, HbA1c hemoglobin A1c, HDLc high density protein, HTN hypertension, LDLc low density protein, SBP systolic blood pressure, TC total cholesterol, TG triglycerides, GFR glomerular filtration rate, UACR urine albumin-to-creatinine Ratio
*Significant P < 0.05; **highly significant P < 0.001
aControl group compared to patients with renal impairment
bControl group compared to patients without renal impairment
cPatients with renal impairment compared to without renal impairment patients
Table 2.
MTHFR genotypes and allele frequencies in the study individuals
| Genotypes/alleles | Control group (n = 200) (%) | HTN patients (n = 200) (%) | OR (95% CI) | P | |
|---|---|---|---|---|---|
| MTHFR C677T | CC | 111 (55.5) | 90 (45) | 1 | Ref. |
| TT | 23 (11.5) | 34 (17) | 1.84 (1.01–3.34) | 0.04 | |
| CT | 66 (33) | 76 (38) | 1.42 (0.92–2.19) | 0.11 | |
| C allele | 288 (72) | 256 (64) | 1 | Ref. | |
| T allele | 112 (28) | 144 (36) | 1.45 (1.07–1.95) | 0.02 |
HTN hypertension, MTHFR methylene tetrahydrofolatereductase
Table 3.
MTHFR genotypes and allele frequencies among hypertensive patients
| Genotypes/alleles | HTN without renal impairment (n = 100) (%) | HTN with renal impairment (n = 100) (%) | OR (95% CI) | P | |
|---|---|---|---|---|---|
| MTHFR C677T | CC | 55 (55) | 35 (35) | 1 | Ref. |
| TT | 13 (13) | 21 (21) | 2.53 (1.13– 5.71) | 0.024 | |
| CT | 32 (32) | 44 (44) | 2.16 (1.16–4.02) | 0.015 | |
| C allele | 142 (71) | 114 (57) | Ref | ||
| T allele | 58 (29) | 86 (43) | 2.02 (1.33–3.08) | < 0.001 |
Table 4.
Multiple regression analysis
| Predictor | Coefficient | P |
|---|---|---|
| Homocysteine | 9.0878 | 0.0011 |
| Cholesterol | 0.0921 | 0.7095 |
| TG | 0.6187 | 0.0608 |
| HDL | − 0.0008 | 0.9994 |
| LDL | 0.6724 | 0.0953 |
| Urea | 1.1749 | 0.1574 |
| Creatinine | 8.402 | 0.591 |
| GFR (ml/min) | − 2.54 | 0.0003 |
| MTHFR (TT genotype) | 0.47 | 0.000 |
Table 5.
Risk factors of renal impairment among different genotypes in hypertension group
| MTHFR C677T | ||||
|---|---|---|---|---|
| CC (n = 90) | CT (n = 76) | TT (n = 34) | P | |
| TC (mg/dl) | 216 ± 26 | 221.5 ± 30 | 258 ± 30b٭ | < 0.01 |
| TG (mg/dl) | 180 ± 30.1 | 190.7 ± 17.6 | 224.1 ± 19.9b٭ | < 0.01 |
| HDLc (mg/dl) | 43.7 ± 9.2 | 40.9 ± 6.8 | 37.4 ± 3.1b٭ | < 0.05 |
| LDLc (mg/dl) | 136.5 ± 30.3 | 141.4 ± 17.9 | 175.1 ± 18b٭ | < 0.05 |
| Urea (mg/dl) | 33.5 ± 10.2a* | 42.9 ± 10.6c٭ | 61.1 ± 13.1b٭ | < 0.01 |
| Creatinine (mg/dl) | 0.89 ± 0.4 | 1.14 ± 0.47 | 2.54 ± 0.84b٭ | < 0.05 |
| GFR (ml/min) | 94.7 ± 10.3a* | 86.7 ± 15.7c٭ | 68.5 ± 11.2b٭ | < 0.01 |
| UACR (mg/gm) | 9.9 ± 2.06a* | 16.67 ± 3.04c٭ | 250.6 ± 51b٭ | < 0.001 |
| Homocysteine (µmol/l) | 13.6 ± 1.06 | 15.1 ± 2.1 | 18.36 ± 1.7b٭ | < 0.05 |
| SBP (mmHg) | 144.7 ± 8.3 | 145.5 ± 6.8 | 156.3 ± 8.7b٭ | < 0.05 |
| DBP (mmHg) | 93.8 ± 4.8 | 95.1 ± 5.7 | 98.13 ± 4.7b٭ | < 0.05 |
DBP diastolic blood pressure, HDLc high density protein, LDLc low density protein, SBP systolic blood pressure, TC total cholesterol, TG triglycerides, UACR urinary albumin/creatinine ratio
aMTHFR CC genotype versus MTHFR CT genotype
bMTHFR CC genotype versus MTHFR TT genotype
cMTHFR CT genotype versus MTHFR TT genotype
Discussion
Hypertension usually results in kidney damage which is an important cause of end stage renal disease, thus it is severely endangering human health [9]. In this study, we investigated the association between MTHFR C677T (rs1801133) gene polymorphism, change on homocysteine level and progression of renal impairment in young age adult hypertensive patients. We showed that there were significant increase of homocysteine levels in hypertensive patients when compared with healthy individuals also there was significant change in homocysteine levels between patients with or without renal impairment on top of hypertension. Folate concentrations showed no changes among all studied individuals. These results were in agreement with Xie et al. [10] who showed that an elevated the plasma Hcy level, even in the range below the cutoff for hyperhomocysteinemia, was a risk factor for renal function and incident renal impairment in a hypertensive population. Hypertension may directly cause renal damage, which then leads to impaired homocysteine excretion and elevated its plasma level, an inverse causality. On the other hand, folate and B12 insufficiency may lead to enzymatic impairment in Hcy metabolic pathways, including remethylation, transsulfuration and adenosylation that may be involved in hypertensive hyperhomocysteinemia [11, 12]. Homocysteine was regarded as an indicator of oxidative stress status. High levels of homocysteine share in oxidative stress status as it increased reactive oxygen species and decreased endothelial nitric oxide cause vascular constriction and stiffness, which could lead to essential hypertension [13]. Increased level of homocysteine affecting the vascular thickness through acceleration of vascular smooth muscle cells proliferation which leads to progression of atherosclerosis and cerebrovascular complications [14, 15]. High concentration of homocysteine increased risk of glomerular sclerosis as it affecting endothelial function of renal blood vessels which plays a critical role in early renal damage in hypertension [16, 17]. We observed that the hypertensive patients had higher levels of lipid profile than healthy individuals while there is no significant change of lipid profile between hypertensive patients with renal impairment and those without renal impairment. We observed that the highest level of UACR was clear hypertensive patients with renal impairment and this similar with previous researcher results [18, 19]. As regards MTHFR C677T, we found that the CC genotype was the most frequent among patients (45%) compared to the other genetic profiles (38% CT and 17% TT). In healthy individuals, the CC genotype was the most frequent (76%) versus 20% and 4% for the CT and TT genetic profiles, respectively. The prevalence of 677T allele was 72% in hypertension patients versus 28% in control group. This was agreed with similar study demonstrated that polymorphism of MTHFR C677T is associated with an increased risk of hypertension, especially elevation T allele frequencies might be increased risk of essential hypertension [20]. The frequency of MTHFR gene polymorphisms varies in different geographical regions, as in African black people the frequency of the TT type homozygote is approximately 1.45% and near 12% in Indians. The frequency of MTHFR C677T mutation is 24% in Jordan and 46.9% in Saudi Arabia people. Among Europeans the prevalence of 677T allele ranged from 24.1 to 64.3%, 2 to 48% among North Americans, 35.5% among Africans and 2% to 63.1% in Asians [21–24]. The prevalence of the mutant T allele of the MTHFR C677T gene in the Chinese population is 41%, which is much higher [25]. Furthermore, we found that hypertensive patients with the MTHFR C677T gene polymorphism showed significantly higher levels of homocysteine and UACR and decreased of creatinine clearance than in the patients with wild type, also dyslipidemia was significantly increased in patients carrying TT genotype compared to wild-type homozygous CC genotype group. Also, hypertensive patients carrying minor T allele were significantly more likely to develop renal impairment compared with those carrying the C allele. The present study suggested that the T allele mutation might be an independent risk factor causing early renal damage in hypertension. Genetic variation of MTHFR enzyme affect Hcy metabolism as MTHFR T allele is related to reduction of MTHFR enzymatic activity which is a critical enzyme in homocysteine remethylation to methionine, decreased blood concentrations of folate and mildly increased plasma homocysteine concentrations [26, 27]. MTHFR activity normally decreased by 60% after 5 min at 46oC, whereas MTHFR mutant encoded by the C677T its activity will decrease by approximately 80 to 90%. This reduction of MTHFR enzyme activity causing the increase of the concentration of blood homocysteine [28]. This relation between MTHFR mutant encoded by the C677T and increased levels of homocysteine may explain the early renal damage in hypertension of young ages. The increased homocysteine produced the change of the glomerular filtration membrane charge selectivity and pore size can cause the presence of urine microalbumin [28]. Many studies had reported that the C677T gene polymorphism is associated with high level of plasma homocysteine, T allele carrier at greater risk for hyperhomocysteinemia compared to C allele and suggested that this variant may be a potential genetic risk factor for cardiovascular diseases [29–31]. However, Pramukarso et al. [32] results were disagreed with these results as they reported that CC alleles associated with the occurrence of hyperhomocysteinemia. Another study found the association of polymorphic C677T gene status and development of cardiovascular disease in rheumatoid arthritis patients as the 677TT genotype is an independent risk factor of increased wall thickness of carotid artery while Hcy was not independent risk factor of increased carotid artery wall thickness [33]. Gnanasambandan et al. [34] reported that CT genotype significantly increased the risk of chronic kidney disease progression in diabetic nephropathy. In the present study, multiple regression analysis showed that 677TT genotype and hyperhomocysteinemia are independent risk factors of increased renal impairment on top of hypertension. The T allele may be considered as a predisposing factor for both elevated Hcy levels and the development of renal impairment in Egyptian hypertensive patients. There is an interaction exists between the 677TT genotype and high blood Hcy concentrations. Many studies suggested a link between genotype mutant homozygotes TT with hyperhomocysteinemia and reported that a T allele carrier at greater risk for hyperhomocysteinemia compared to C allele [35–38]. On the contrary, one study reported that CC alleles associated with the occurrence of hyperhomocysteinemia [32].
Conclusion
Genetic variants of C677T MTHFR gene and hyperhomocysteinemia may be responsible for rapid progress of renal impairment in Egyptian young age hypertensive patients. TT genotype or T allele may be considered as a predisposing factor for both elevated Hcy levels and the development of renal impairment. This study believed that lowering of homocysteine level can reduce renal impairment of hypertensive patients.
Acknowledgements
The authors wish to thank all participants of this study.
Author Contributions
All authors share in design of the study, Khaled A Elhefnawy collected the patients, gathered the clinical data. Hanaa H. Elsaid and Saffa M. Elalawi did the laboratory work of the study. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published.
Funding
No sources of funding except authors.
Availability of Data and Materials
All data generated or analysed during this study are included in this published article.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no competing interests.
Consent for Publication
Not applicable.
Ethics Approval and Consent to Participate
The ethical approval was taken from the Zagazig University Ethics Committee guidelines in Egypt according the Declaration of Helsinki.
Footnotes
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Contributor Information
Hanaa H. Elsaid, Email: hanaahosny52@yahoo.com
Khaled A. El-Hefnawy, Email: kelhefnawy@gmail.com
Saffaa M. Elalawi, Email: saffaaalawi@yahoo.com
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Data Availability Statement
All data generated or analysed during this study are included in this published article.