NOS3 gene intron 4 a/b polymorphism is associated with ESRD in autosomal dominant polycystic kidney disease patients

Abstract Introduction: Endothelial nitric oxide synthase (eNOS) genes have been implicated in renal hemodynamics as potent regulators of vascular tone and blood pressure. It has been linked to a reduction in plasma nitric oxide levels. Several studies have recently been conducted to investigate the role of NOS3 gene polymorphisms and end-stage renal disease (ESRD). However, the results are still unclear and the mechanisms are not fully defined. As a result, we conducted a meta-analysis to examine the relationship between NOS3 gene polymorphism and ESRD in autosomal polycystic kidney disease (ADPKD) patients. Methods: To assess the relationship between NOS3 gene polymorphism and ESRD, relevant studies published between September 2002 and December 2020 were retrieved from the PubMed (Medline), EMBASE, Google Scholar, and Web of Science databases. The pooled odds ratio (OR) and 95 % confidence interval (CI) were calculated using a fixed-effect model. To assess the heterogeneity of studies, we used Cochrane’s Q test and the Higgins and Thompson I2 statistics. Results: Our meta-analysis of 13 studies showed that the presence of the two NOS3 gene polymorphisms significantly increased ESRD risk in ADPKD patients with 4a/b gene polymorphism (aa+ab vs. bb: OR=1.95, 95% CI=1.24-3.09, p=0.004). In addition, no significant association was found between the NOS3 894G>T (Glu298Asp) polymorphism and the risk of ESRD in ADPKD patients (GT+TT vs. GG: OR=1.21, 95% CI=0.93-1.58, p=0.157). There was no evidence of publication bias. Conclusions: The findings of the current meta-analysis suggest that NOS3 intron 4a/b polymorphism plays a vital role in the increasing risk of ESRD in ADPKD patients.

Introduction: Endothelial nitric oxide synthase (eNOS) genes have been implicated in renal hemodynamics as potent regulators of vascular tone and blood pressure. It has been linked to a reduction in plasma nitric oxide levels. Several studies have recently been conducted to investigate the role of NOS3 gene polymorphisms and end-stage renal disease (ESRD). However, the results are still unclear and the mechanisms are not fully defined. As a result, we conducted a meta-analysis to examine the relationship between NOS3 gene polymorphism and ESRD in autosomal polycystic kidney disease (ADPKD) patients. Methods: To assess the relationship between NOS3 gene polymorphism and ESRD, relevant studies published between September 2002 and December 2020 were retrieved from the PubMed (Medline), EMBASE, Google Scholar, and Web of Science databases. The pooled odds ratio (OR) and 95 % confidence interval (CI) were calculated using a fixedeffect model. To assess the heterogeneity of studies, we used Cochrane's Q test and the Higgins and Thompson I 2 statistics. Results: Our meta-analysis of 13 studies showed that the presence of the two NOS3 gene polymorphisms significantly increased ESRD risk in ADPKD patients with 4a/b gene polymorphism (aa+ab vs. bb: OR=1.95, 95% CI=1.24-3.09, p=0.004). In addition, no significant association was found between the NOS3 894G>T (Glu298Asp) polymorphism and the risk of ESRD in ADPKD patients (GT+TT vs. GG: OR=1.21, 95% CI=0.93-1.58, p=0.157). There was no evidence of publication bias.

Conclusions:
The findings of the current meta-analysis suggest that NOS3 intron 4a/b polymorphism plays a vital role in the increasing risk of ESRD in ADPKD patients.

IntroductIon
Renal cysts can have various etiologies, broadly classified into genetic and non-genetic disorders. The most common and widely accepted genetic cause of renal cystic disease in humans is autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and juvenile nephronophthisis, for which genes or chromosomal locations have been identified 1 . Among all renal cystic diseases, ADPKD is genetically heterogeneous and affects all racial groups worldwide, associated with liver, cardiovascular, gastrointestinal and genital abnormalities, with an estimated frequency between 1:400 and 1:1,000 2 . ADPKD may be caused by mutations in one of the two genes, namely polycystic kidney disease 1 (PKD1), mapped to 16p13.3, and polycystic kidney disease 2 (PKD2) gene on chromosome 4q21 3 . The frequency of mutations of PKD1gene is much higher and the gene is responsible for 85% of ADPKD cases, while 15% is caused by the PKD2 gene. Additionally, elderly patients have more cases of PKD2 mutations than of PKD1 mutations. Renal disease involves hypertension, urinary tract infections (UTI), hematuria, renal pain, and renal insufficiency, and end-stage renal disease (ESRD) occurs in approximately 50% of ADPKD patients in their late forties 4 .
ESRD is a multifactorial disease and has been shown to be more significant in many aspects of this genetic factor. Previous evidence has proven that impaired nitric oxide synthase 3 (NOS3) contributes to vascular endothelial dysfunction, and blood endothelial cells are suggested to play a vital role in the pathogenesis of ESRD 5 . NOS3 is a dimeric cellular signaling molecule with significant regulatory activities such as glomerular vasodilatation, which is essential for controlling the glomerular filtration rate (GFR). In this perspective, NOS3 in ADPKD was considered a possible candidate gene for ESRD 6 . The NOS3 gene was mapped to a chromosome of 7q36 consisting of 26 exons. Several polymorphic variants have been associated to modified NO synthesis, including promoter -786 T > C, 894 G > T, and intron 4 variable tandem repeats a / b (VNTR) polymorphisms. Among these, the 27-base pair (bp) (VNTR) intron-4 of NOS3 is known to alter eNOS expression and cause impaired NO synthesis 7 . The genotypes and haplotypes of NOS3 tagSNPs were not associated with the disease 6 .
Although many researchers examined the association between NOS3 gene polymorphisms and ESRD in ADPKD, the findings were inconsistent [7][8][9][10][11][12] . In view of the clinical heterogeneity of ADPKD, this study aimed to quantitatively summarize the association between NOS3 polymorphisms (894G>T intron 4 VNTR a / b polymorphism) and ESRD risk in ADPKD by conducting a comprehensive metaanalysis of all eligible case-control studies.

identificAtion of eligibility studies
Articles published between September 2002 to December 2020 on the associations of the NOS3 gene polymorphism and ESRD were identified. All casecontrol studies considering the association in ADPKD patients published in English languages were selected and organized according to the PRISMA guidelines 13 .

inclusion And exclusion cRiteRiA
To conduct a more robust meta-analysis, two authors collected data from all relevant articles independently. Our selection criteria were: (1) case-control studies on the association between NOS3 polymorphisms and risk of ESRD in ADPKD, (2) available fulltext articles, (3) written in English, and (4) original data and complete genotype allele count for both the case and control groups were available. Studies were excluded if they (1) had overlapping/duplicate data, (2) had no control group, (3) did not have clear genotype data, and (4) were case reports and review articles. All information about the selection of studies in ADPKD patients with or without ESRD was arranged.

stAtisticAl AnAlysis
The genotype data of case and control groups were recorded. The comparison group already included a selection, but NOS3 genotypes were not tested for Hardy-Weinberg equilibrium (HWE).
The meta-analysis was carried out using the web tool MetaGenyo. For each study, the strength of association between ESRD risk in ADPKD and NOS3 gene polymorphisms (894G>T and intron 4 VNTR) was assessed by the summary odds ratio (OR) and the 95% confidence interval (CI) in the dominant model. To assess the between-study heterogeneity, we used Q and I 2 statistics in all studies. To assess the robustness of findings, sensitivity analysis was performed using a "leave-one-out" meta-analysis. Egger's test and Begg's funnel plot were used to assess publication bias. All statistical analyses were done using the Comprehensive Meta-analysis software. A P-value of <0.05 was considered statistically significant.

chARActeRistics of the studies
Using the aforementioned search strategy, 82 articles were identified. From these, 22 duplicate and irrelevant articles were excluded. After reading the abstracts and titles, 31 articles that did not assess the association between NOS3 894G>T and intron 4a/b polymorphisms and ESRD risk were excluded. Twenty-nine articles were fully reviewed, of which 9 papers with 520 ADPKD patients with ESRD and 563 ADPKD patients without ESRD for the NOS3 894G>T polymorphism 8,10-12,14-18 and 5 papers with 185 ADPKD patients with ESRD and 223 ADPKD patients without ESRD for NOS3 intron 4a/b polymorphism 7,9,11,19,20 that had sufficient data were included in the present meta-analysis. The process of selecting papers is depicted in Figure 1. The characteristics of all included studies are listed in Table 1.

MetA-AnAlysis of NOS3 gene polyMoRphisMs And esRd Risk in Adpkd
The association between NOS3 polymorphism variants and ESRD risk in ADPKD was assessed in a dominant model ( Figure 2). Overall, the pooled analyses showed that the eNOS 4a/b polymorphism is significantly associated with increased risk of ESRD in the fixed-effect model (aa+ab vs. bb: OR=1.95, 95% CI=1.24-3.09, p=0.004) ( Figure 2B). However, there was no significant association between NOS3 894G>T polymorphism and the risk of ESRD in ADPKD

test of heteRogeneity, sensitivity, And publicAtion biAs
The estimated effect sizes for both NOS3 gene polymorphisms (894G>T: I 2 = 11.3 %, pheterogeneity =0.341; and intron 4a/b polymorphism: I 2 = 15.6 %, pheterogeneity =0.315) showed significant heterogeneity. Sensitivity analyses were performed by excluding studies one at a time and conducted the analysis after each omission. There were no statistically significant differences in polymorphism data, indicating that the analysis was statistically reliable and consistent ( Figure 3A).
The analysis of the intron 4a/b polymorphism in two studies 19,20 demonstrated that the pooled ORs increased when each study was omitted ( Figure 3B). Begg's funnel plot and Egger's test were used to assess the publication bias of the literature. The shape of the funnel plot was asymmetric for both the 894G>T and intron 4a/b polymorphisms. In support of this, the Egger test revealed no evidence of significant publication bias ( Figure 4A and 4B). Furthermore, Egger's linear regression test also revealed no publication bias for studies on the 894G>T (P = 0.915) and intron 4 a/b polymorphisms (P = 0.159).

dIscussIon
We included 13 published studies in this metaanalysis that revealed that NOS3 4a/b polymorphisms were significantly associated with various vascular complications, which are a cause of ESRD in ADPKD patients. Our study also showed that there is no heterogeneity or publication bias in the included studies. However, the results of the sensitivity analysis in each study group indicated that the pooled OR estimates were not changed quantitatively after each omission. Although the study suggests that nitric oxide may play a role in ADPKD pathophysiology, the NOS3 894G>T polymorphism failed to demonstrate an association with susceptibility to ESRD in ADPKD patients. The findings are consistent with a previous meta-analysis that found that the NOS3 intron 4a/b polymorphism increased the risk of ESRD in ADPKD patients 21 .
The NO synthases (NOS) are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine in vascular endothelial cells 22 . It is well known that NO is highly reactive due    eNOS gene expression is independently predictive of hypertension in the ADPKD population 27 .

894G>T (Glu298Asp) Polymorphism
In kidney disease, NO production is reduced by either a decrease in the enzyme substrate (L-arginine) or an increase in the bioavailability of the enzyme inhibitor asymmetric dimethylarginine (ADMA), which in turn reduces NO synthesis via a feedback mechanism 28 . This mechanism has been shown to accelerate the progression of pre-existing kidney disease. Various studies have shown that NO negatively regulates the renin-angiotensin system by inhibiting ACE activity and AT1 receptors 29 . The release of NO by endothelial cells plays a major role in regulating the local hemodynamics and systematic blood pressure 30 . Decreased production of NO plays a major role in the progression of renal disease 28 . A significant decrease in different isoforms of NOS in the cystic epithelium was observed during the growth of a renal cyst in Han: Sprague-Dawley (SPRD) polycystic rats 31 . Several lines of evidence suggest that ADPKD is characterized by endothelial dysfunction caused by impaired NO release 32,33 .
Animal models and clinical trials have demonstrated the importance of NOS in polycystic kidney disease 34 . ADPKD patients with the 4a allele progressed to ESRD more slowly in Belgium and France, whereas Hellen's patients from Greece and Cyprus progressed faster 35,36 . However, some studies suggested that this locus was not linked with ESRD of different etiologies 36,37 . The substitution of aspartic acid for glutamate affects the domain of the oxidase enzyme that serves as a binding site for BH4 and the amino acid L-arginine. The change causes an enzyme variation, making it more susceptible to proteolytic cleavage in position D238-P239. Further, it produces a shorter form of the enzyme, resulting in less NO production 38 . However, the relationship between 894G>T polymorphism and the age of onset of ESRD in ADPKD patients also yielded inconsistent results 15,16,37 . While the -786T>C (rs2070744) polymorphism has a functional effect, it is linked to the replication protein A1 (RPA1), which binds to the NOS3 promoter with high affinity when the C allele is present, resulting in reduced NOS3 transcription 39 .
No significant findings were observed regarding the promoter -786 T>C polymorphism with ESRD progression in patients with type 1 ADPKD 35,40 . Our findings were inconclusive in terms of ethnicity-specific associations between NOS3 gene polymorphisms and to its short half-life and potent regulator of vascular tone and hemorheology via the activation of the cyclic guanosine monophosphate (cGMP)-dependent pathway 23 . Besides, it has directly involved in the vascular endothelium, complex cellular interactions, and global inflammation-mediated cell activation 24 . Generally, NO inhibits NaCl absorption along the nephron. Several studies have shown that NOS inhibitors such as nitro-L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) have a tonic influence, especially on medullary circulation 25 . Despite this, renal NO synthesis is involved in the acute and chronic regulation of sodium balance.
Hypertension is the most frequent complication in ADPKD patients, occurring in approximately 60% of the patients. Miyamoto et al. discovered that the 894G>T (Glu298Asp) missense variant was significantly associated with essential hypertension, suggesting a genetic susceptibility for essential hypertension 26 . Another study found that eNOS expression and eGFR were significantly higher in ADPKD patients without hypertension than in those with hypertension. This demonstrates that ESRD in ADPKD patients. This is possibly due to differences in the allele frequencies of the NOS3 gene among populations. Individual studies investigating the link between NOS3 polymorphisms and ESRD complications have also been conducted. Still, these studies may have been imbalanced due to the inability of individual components to identify the desired impact of these polymorphisms.
Certain limitations and biases of the study have to be considered and resutls should be interpreted with caution. Our meta-analysis indicated that the NOS3 intron 4a/b polymorphism, but not the G894T polymorphism, seems to increase the risk of ESRD in ADPKD patients. More high-quality studies are needed to investigate the complexities of the associations between NOS3 gene polymorphisms and the therapeutic implications of ESRD in ADPKD patients.

authors' contrIbutIon
LVKSB contributed to the conception or design of the study. UNP, conducted methodological search and data collection. UNP analyzed the data. UNP, MM, LS, and HKV prepared primary manuscript. LVKSB conducted critical review and finalized the manuscript. All the authors read and approved the final manuscript.