Evaluation of common polymorphisms of eNOS gene and ACE gene in autosomal dominant polycystic kidney disease patients and their association with HTN and renal failure

Implication for health policy/practice/research/medical education: In a study on 75 ADPKD individuals and 100 control subjects, we found, the frequency of DD polymorphism of ACE gene in ADPKD patients was meaningfully higher than that in the control group; however the polymorphism of this gene was not significantly different in patients with and without hypertension or renal insufficiency in ADPKD patients. Therefore, despite the increased activity of the renin-angiotensin-aldosterone system in these patients, it seems that the ACE gene polymorphism is not able to determine the progression of blood pressure and renal disease in these patients. However, more studies are necessary on this subject. Please cite this paper as: Malakoutian T, Madadi B, Ebrahimi A. Evaluation of common polymorphisms of eNOS gene and ACE gene in autosomal dominant polycystic kidney disease patients and their association with HTN and renal failure. J Renal Inj Prev. 2020; 9(1): x-x. doi: 10.15171/jrip.2020.xx. O rig in al


Introduction
Autosomal dominant polycystic kidney disease (ADPKD) is a late-onset systemic disorder that presents with bilateral renal cysts, cysts in other organs such as the liver, seminal vesicles, pancreas and arachnoid membranes, vascular abnormalities including intracranial aneurysms, aortic root dilatation and thoracic aortic dissection, mitral valve prolapse, and also herniation of the abdominal wall. The renal manifestations include hypertension, renal pain, and renal failure (1). ADPKD is the most common hereditary renal disease, with an estimated prevalence of approximately 1: 1000 (2,3). Although ADPKD is a hereditary illness, at least 10% of families have new mutations (1).
Approximately 50% of patients with ADPKD have endstage renal disease (ESRD) of up to 60 years of age (1,4,5), accounting for about 8%-10% of patients undergoing renal replacement therapy (2). On the other hand, the early onset of hypertension is a major manifestation in these patients, with 60% of them having hypertension before decrease of glomerular filtration rate (GFR) (6). In addition, high blood pressure is an important parameter in the development of chronic renal failure (CRF) in these patients (3,(6)(7)(8). Some studies have suggested that hypertension causes cysts to grow faster and thus can rupture in cases such as intracranial aneurysms. Therefore, early treatment of hypertension can be associated with reduced morbidity and mortality in individuals with ADPKD (4).
The pathogenesis of high blood pressure and the progression of kidney disease in ADPKD patients is related with increased kidney volume and cyst density in both children and adults (9). Recent studies have shown that renal function remains relatively stable as long as the kidney volume is less than 1500 cubic centimeters, while a rapid decline in renal function can lead to renal replacement therapy. It is believed that distortion of renal structure could lead to tubular structural destruction and dysfunction, resulting in the activation of the reninangiotensin-aldosterone (RASS) system, which increases blood pressure prematurely (4,5,7,(10)(11)(12). Therefore, RAAS antagonists can prevent cell proliferation and inflammation and control hypertension in these patients (11,12). In addition, it has been reported that the prevalence of hypertension in the general population differs in the polymorphisms of DD, DI and II of the ACE gene (13).
Additionally, release of nitric oxide (NO) by endothelial cells plays an important role in the control of local hemodynamics and systemic hypertension. Accordingly, the disturbance in the production of NO in endothelial cells is an important finding for defective vasodilatation and thus hypertension in ADPKD patients (14). In these patients, there is also an impaired endothelial-dependent vasodilation, which can be an early and strong marker for progression of renal disease (6,15). This damage results from the reduction of NO production by endothelial nitric oxide synthase (eNOS) (16) which is encoded by the eNOS gene (17). Therefore, the eNOS gene polymorphisms can alter renal vascular endothelial function, creating kidney failure and hypertension in these patients by effect on NO production (6).
Although high blood pressure and kidney failure in ADPKD individuals require prediction and precise monitoring, the factors associated with the severity of renal disease in these patients have not yet been identified and there is not a good indicator for predicting the clinical course of the disease and its outcomes. Various studies have reported different intra-familial and inter-familial phenotype variations of ADPKD that indicate involvement of both environmental and genetic factors in the disease (18). On the other hand, the polymorphism of the ACE and eNOS genes is different in various populations since it is associated with high blood pressure (19), which may be helpful in predicting the clinical course of these patients (20). However, the relationship between the abovementioned gene polymorphisms with high blood pressure and kidney failure in ADPKD patients has been different in many studies (21) and is still controversy. Thus, the aim of this study was to evaluate the association between the common polymorphisms of eNOS and ACE genes with clinical manifestations (high blood pressure and kidney failure) in these patients.

Study protocol
This case-control study was performed on 75 patients with ADPKD over 18 years of age who referred to Hasheminejad hospital in Tehran (2015 to 2016). Diagnosis of ADPKD was based on patient family history and kidney ultrasound. In addition, 100 attendants of patients who did not mention a specific problem were randomly selected as the control group. First, the study design was explained to all participants and a written consent form received from all of them. Then, participants' data including age, gender, weight, height, BMI (body mass index), history of hypertension (the participants who had a history of taking blood pressure pills), presence or absence of diabetes and IHD (ischemic heart disease), and serum creatinine level were recorded using a checklist. Their blood pressure was taken at least 2 separate visits in Hasheminejad clinic and BP ≥140/90 mm Hg was considered hypertensive. GFR was measured by Cockcroft-Gault formula. None of them had history of IHD. They had not any rise in their serum creatinine level at least for the past 3 months. GFR less than 60 mL/min was considered CRF, since GFR more than 60 mL/min was considered normal. Subsequently, 7.5 cc of whole blood sample was received from each participant which 5 cc inserted in EDTA solution and 2.5 cc in RNA later or TRIzol solution. The samples were refrigerated at -70°C until sending to the laboratory. Specimens were sent to the genetic laboratory for genetic evaluation. In the laboratory, DNA was extracted using phenol chloroform solution and ethanol deposition techniques (9). Then, genotyping was performed for the I/D polymorphism of ACE gene and Glu298 ASP and T786C polymorphisms of the eNOS gene using PCR electrophoresis and molecular evaluation with specialized primers of these two set genes ( Figure 1A-1C).

Ethical approval
The research followed the tenets of the Declaration of Helsinki. The ethics committee of Iran University of Medical Sciences approved this study (IR. IUMS. REC1395.9311402002). Accordingly, written informed consent was taken from all participants before any intervention. This study is extracted from the nephrology fellowship thesis of Bahareh Madadi at this university.

Statistical analysis
SPSS Statistics for Windows, Version 25.0 (IBM Corp, Armonk, N.Y., USA) was used to analyze the data. Frequency and percentage as well as mean and standard deviation were used to describe the variables. Chi-square test and independent samples t test were used for data analysis and comparison between groups, respectively. A P value of less than 0.05 was considered statistically significant.

Results
Forty-five patients (60%) in the ADPKD group and 48 participants (48%) in the control group were female. The mean (SD) of age of ADPKD and control groups was 44 ± 15 years and 41 ± 11 years, respectively. The two groups did not have a significant difference in age and gender. There was no significant difference between the two groups in terms of height, weight and BMI too ( Table 1). In the ADPKD group, 46 patients (61%) had hypertension and 5 (6.7%) had diabetes, while none of the control people were hypertensive and had diabetes (Table  1). Forty-two patients (56%) had GFR <60 mL/min/1.73 m 2 in the ADPKD group (Table 1). Table 2 compares the polymorphisms of ACE and eNOS genes between ADPKD and control groups. In the case of ACE gene, the most common polymorphism in the ADPKD group was DD (43%), while in the control group was ID (53%), which was a significant difference between the two groups (P < 0.001). For the T786C polymorphism of the eNOS gene, the most common type in the ADPKD type was TC (57%), whereas in the control group was TT (68%), which also had a significant difference between the groups (P < 0.001). However, in both ADPKD and control groups, the most common type of Glu298 ASP polymorphism of eNOS gene was GG (56% and 61%, respectively), with no significant difference between the two groups was detected (P = 0.311).
Comparing the different characteristics of the two groups of ADPKD patients with and without hypertension, we found that hypertensive ADPKD patients were more male, had higher age and serum creatinine level, and also they had more GFR <60 mL/min/1.73 m 2 (74% versus 28%) ( Table 3). However, the polymorphism of ACE and eNOS genes did not differ significantly between them (Table  4 and Figure 2A-2C). In addition, the polymorphism of these genes in ADPKD patients with and without decreased GFR was not significantly different (Table 5).   renal failure than the control group. We also found that the polymorphism of the ACE gene and the T786C polymorphism of the eNOS gene differed significantly in ADPKD patients with the control group but the Glu298 ASP polymorphism of the eNOS gene did not differ with the control group. Additionally, hypertensive ADPKD patients were more male, with a higher age, and had significant renal insufficiency. However, the polymorphisms of ACE and eNOS genes between ADPKD patients with and without hypertension and also in patients with and without decreased GFR were not significantly different. Therefore, the polymorphism of the ACE and eNOS genes can help in the diagnosis of ADPKD patients, however, it seems that it is not a helpful marker in predicting the hypertension status and severity of kidney disease in these patients. Several studies have shown, an endothelial dysfunction due to NO production disturbances in ADPKD patients (22). Similarly, human and animal models of this disease have confirmed changes in endothelium-dependent vasoconstriction (16). However, the results of various studies on the relationship between the severity of kidney disease or hypertension in ADPKD patients and the eNOS gene polymorphism are different. Persu et al assessed the

Discussion
The findings of this study showed that patients with ADPKD were more likely to have hypertension and effect of the Glu298Asp polymorphism on the onset age of renal failure in ADPKD patients and concluded that it was accompanied by a 5-year decrease in mean age of renal failure in male patients. This condition could be due to decreased NOS activity, thereby reducing the endothelial production of NO (17). However, this effect was not observed in women, which is said to be due to the ability to stimulate endothelial NO production by estrogen (22). Tazon-Vega et al reported that NOS3 polymorphisms have little involvement in the ADPKD renal outcomes (8). On the other hand, Stefanakis et al reported that in ADPKD individuals, the Glu298Asp polymorphism of the NOS3 gene was associated with the onset of ESRD and the T allele was associated with an earlier progression to ESRD (23). Dasar et al showed that the prevalence of GG, GT and TT genotypes of NOS3 gene in ADPKD patients with kidney failure were 66.7%, 33.3% and 0%, respectively, in ADPKD patients without kidney failure were 78.6%, 19%, 2.4%, and in control group were 64.3%, 35.7% and 0% respectively, while there was no significant difference between the groups. In addition, there was no significant relationship between Glu298Asp polymorphism and the onset age of ESRD (24). Ramanathan et al also showed that NOS3 tag SNPs do not play a role in the progression of renal failure in ADPKD patients (25).
In a review article about relationship of the eNOS gene polymorphism with the progression of renal disease in ADPKD patients, Xue et al reported that the GG genotype of the Glu298Asp variant reduces the progression of renal failure. However, there was no positive relationship of this genotype with hypertension or presence of renal failure (6). Kocyigit et al also reported that, the expression of the eNOS gene is independently predictive of hypertension in ADPKD patients, while there is no link between eNOS gene polymorphism and hypertension in these patients (20). The relationship between other eNOS gene polymorphisms and renal failure in ADPKD patients has also been reported (26)(27)(28). Our study showed that the T786C polymorphism of the eNOS gene in ADPKD patients was different with the control group, but the Glu298 ASP polymorphism did not differ with the control group. Additionally, eNOS gene polymorphism was not significantly different between ADPKD patients with and without hypertension and also in patients with and without GFR reduction. Differences in the results of the above studies may indicate differences in the studied populations and differences in the method of study.
Since, it seems that, the commonly occurring Glu298ASP polymorphism of the eNOS gene, is associated with high blood pressure in the general population, however, is not able to predict the course of disease and hypertension in ADPKD patients.
On the other hand, it has been shown that the DD genotype of ACE gene in humans is associated with higher serum levels of ACE and hypertension (29,30). O'Donnell et al also reported in the Framingham heart study participants, that the polymorphism of the ACE gene is related only to men with hypertension and diastolic blood pressure (13). Baboolal et al (31) and Pérez-Oller et al (32) reported that the DD genotype of the ACE gene was associated with the progression of renal failure in ADPKD patients. Tazon-Vega et al also reported that ACE polymorphism has a brief effect on the onset age of renal failure in these patients (8). However, in other studies, no relationship between ACE polymorphism and hypertension or kidney disease course was detected. Likewise, Lee et al found no association between the I/D polymorphism of ACE gene and hypertension or renal failure in ADPKD patients (33). Similarly, Schiavello et al also failed to establish a relationship between the ACE gene polymorphism with the severity of the ADPKD phenotype in several different races (34). Ecder et al failed to establish a relationship between ACE gene polymorphism and hypertension in these patients (35) and Metra et al also found no association between ACE gene polymorphisms and kidney disease progression (36). Pereira et al in a meta-analysis of 13 studies showed that the ACE gene polymorphism does not affect the progression of renal disease in ADPKD patients (37). Kocyigit et al also reported no association between ACE gene polymorphism and hypertension in ADPKD patients (20). Our study also showed that, the frequency of DD polymorphism of ACE gene in ADPKD patients was significantly higher than that in the control group, however the polymorphism of this gene was not significantly different in patients with and without hypertension or renal insufficiency. Therefore, despite the increased activity of the renin-angiotensinaldosterone system in these patients, it seems that, the ACE gene polymorphism is not able to determine the progression of blood pressure and renal disease in these patients.
Finally, we can conclude that current studies on the gene polymorphism of the renin-angiotensin-aldosterone system and its role in kidney failure and hypertension in ADPKD patients have been performed in different populations with different designs. Hence, the results of various studies are different. Additionally, it appears that more proportion of patients is necessary to investigate the relationship of polymorphisms and manifestations of ADPKD. However, considering the results of the above studies, it can be concluded that, currently no specific polymorphism that can predict the course of the disease in ADPKD patients was existed.

Conclusion
The findings of this study showed that the polymorphism of the ACE gene and the T786C polymorphism of the eNOS gene differed in ADPKD patients with the control group, but the Glu298 ASP polymorphism of the eNOS gene was not different from the control group. However, the polymorphism of ACE and eNOS genes between was not significantly different in ADPKD patients with and without hypertension or decreased GFR. Therefore, although the polymorphism of the ACE and eNOS genes can help in the diagnosis of ADPKD patients, it seems that it is not a helpful marker for predicting the course of the disease. However, more studies are necessary on this subject.

Limitations of the study
Our investigation was conducted on a limited sample size. Accordingly, additional investigations should be carried out on larger population regarding this subject.
Authors' contribution TM and BM; data collection and manuscript drafting. AE; genetic study. All authors read and signed the final paper.

Conflicts of interest
The authors declare no conflict of interest.

Ethical considerations
Ethical issues (including plagiarism, misconduct, data fabrication, falsification, double publication or submission, redundancy) have been completely observed by the authors.

Funding/Support
This study was supported by a grant from Iran University of Medical Sciences.