Association between Dietary Intakes of Nitrate and Nitrite and the Risk of Hypertension and Chronic Kidney Disease: Tehran Lipid and Glucose Study

Background and Aim: The association of habitual intakes of dietary nitrate (NO3−) and nitrite (NO2−) with blood pressure and renal function is not clear. Here, we investigated a potential effect of dietary NO3− and NO2− on the occurrence of hypertension (HTN) and chronic kidney disease (CKD). Methods: A total of 2799 Iranian adults aged ≥20 years, participating in the Tehran Lipid and Glucose Study (TLGS), were included and followed for a median of 5.8 years. Dietary intakes of NO3− and NO2− were estimated using a semi-quantitative food frequency questionnaire. Demographics, anthropometrics, blood pressure and biochemical variables were evaluated at baseline and during follow-up examinations. To identify the odds ratio (OR) and 95% confidence interval (CI) of HTN and CKD across tertile categories of residual energy-adjusted NO3− and NO2− intakes, multivariate logistic regression models were used. Results: Dietary intake of NO3− had no significant association with the risk of HTN or CKD. Compared to the lowest tertile category (median intake < 6.04 mg/day), the highest intake (median intake ≥ 12.7 mg/day) of dietary NO2− was accompanied with a significant reduced risk of HTN, in the fully adjusted model (OR = 0.58, 95% CI = 0.33–0.98; p for trend = 0.054). The highest compared to the lowest tertile of dietary NO2− was also accompanied with a reduced risk of CKD (OR = 0.50, 95% CI = 0.24–0.89, p for trend = 0.07). Conclusion: Our findings indicated that higher intakes of NO2− might be an independent dietary protective factor against the development of HTN and CKD, which are major risk factors for adverse cardiovascular events.


Introduction
Elevated blood pressure and renal dysfunction represent world-wide public health problems and are known to be major underlying causes for cardiovascular disease morbidity and mortality [1,2].
The key role of disrupted nitric oxide (NO) pathway, including either decreased production or reduced bioavailability of NO, is now well established in the pathogenesis of hypertension (HTN) and kidney disease [3][4][5]. The interaction of NO pathway with cardiorenal disease involves alterations of the renin-angiotensin (ANG) system, eicosanoid pathways, endothelines, cytokines, and regulators of inflammatory pathways [6]. NO deficiency has been associated with glomerular HTN and ischemia, glomerulosclerosis, proteinuria and kidney dysfunction [4,7,8].
In recent years, following the discovery of potential ability of inorganic nitrate (NO 3 − ) and nitrite (NO 2 − ) as an important back-up system for impaired NO synthase (NOS)-derived NO generation, the historical conception of the scientific community focused on the potential hazards of NO 3 − and NO 2 − exposures [9,10] shifted towards therapeutic properties of these compounds in cardiometabolic disorders [11][12][13][14][15][16][17][18][19][20][21]. Theoretically, reductions of NO 3 − and NO 2 − to NO could restore NO homeostasis, maintain the steady-state NO levels, and are considered as stable storage pools for NO-like bioactivity [22]. So, considering the role of NO as the key regulator of vascular homeostasis and natural vasodilator, supplementation with inorganic NO 3 − and NO 2 − have been investigated as potential therapeutic options in cardiovascular disease, including HTN, and in states renal dysfunction [23][24][25][26]. Currently, a large body of evidence supports a crucial role of NO 3 − and NO 2 − in the regulation and modulation of blood flow, endothelial function, and blood pressure [26][27][28].
Pre-clinical studies also confirm protective effects of NO 3 − and NO 2 − against ischemia-reperfusion injury, arterial stiffness, oxidative stress, inflammation and intimal thickness [26]. However, the nutritional aspects of the vasculo-protective effects of these anions are not clear and their long-term effects are still unknown; there is therefore a critical importance for good evidence to clarify the endpoints in the framework of epidemiological studies [29]. Following our findings regarding the protective effect of NO 3 − -containing vegetables against development of HTN, we speculated that the observed effect may be related to NO 3 − [30]; after development of a valid database of NO 3 − /NO 2 − content of food items [31], we expanded our hypothesis in the framework of the current study to clarify potential effects of NO 3 − /NO 2 − on the risk of HTN and CKD.
To the best of our knowledge, the potential impact of dietary NO 3 − and NO 2 − on the occurrence of HTN and renal dysfunction has not yet been investigated in prospective longitudinal examination; such a setting could probably help to better justify the abovementioned experimental and clinical findings and provide more practical data for dietary recommendations regarding NO 3 − and NO 2 − .
The main focus in this study, therefore, was to ascertain whether regular intake of NO 3 − and NO 2 − could predict the occurrence of HTN and chronic kidney disease (CKD) among an Iranian population, during a 6-year follow-up.

Study Population
This study was conducted within the framework of the Tehran Lipid and Glucose Study (TLGS), an ongoing community-based prospective study being conducted to investigate and prevent non-communicable diseases, in a representative sample in the district 13 of Tehran, the capital city of Iran [32]. During the third phase of the TLGS (2006-2008), a total of 12,523 subjects completed the examinations, of which 4920 were randomly selected for completing the dietary assessment based on their age and sex. The randomization was performed because of cost and complexity of dietary data collection in large populations and also the fact that this process is time consuming. Finally, the dietary data for 3462 subjects who agreed to participate and completed the food frequency questionnaire (FFQ) were available. The characteristics of participants who completed the validated FFQ were similar to those of the total population in the third phase of TLGS [33]. For the current analysis of, 2799 adult men and women (≥20 years) with complete data (demographics, anthropometrics, biochemicals and dietary data), were recruited. Two separate lines of exclusions were carried out for HTN and CKD as the outcomes. First, for the analysis of incident HTN, after exclusion of the participants with prevalent HTN at baseline (n = 372), and the participants with under-or over-reported energy intakes (<800 or ≥4200 kcal/day) or specific diet (including dietary recommendations for HTN, hyperlipidemia or diabetes) (n = 300), the remaining subjects were followed up to the fourth (2009-2011) and fifth TLGS (2012-2014) examinations. Participants who had no follow-up data (n = 249) were also excluded and final analyses was conducted on 1878 adults (806 men and 1072 women). Second, for the analysis of incident CKD, after exclusions included individuals with prevalent CKD at baseline (n = 487), unusual diet (n = 237), along with 295 who did not attend any follow-up examinations, resulting in a total number of 1780 adults (727 men and 1053 women). A flowchart of the study population is shown in Figure 1. Written informed consents were obtained from all participants, and the study protocol was approved by the ethics research council of the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences in Teheran (18ECRIES93/11/26).

Demographic, Anthropometric and Clinical Measures
Trained interviewers collected information using standard questionnaires. Detailed measurements of variables in TLGS have been reported elsewhere [32]. Smoking status was obtained using face-to-face interviews; subjects who smoked daily or occasionally were considered current smokers. Weight was measured to the nearest 100 g using digital scales, while the subjects were minimally clothed, without shoes. Height was measured to the nearest 0.5 cm, in a standing position without shoes, using a tape meter. Body mass index (BMI) was calculated as weight (kg) divided by square of the height (m 2 ). Waist circumference (WC) was measured to the nearest 0.1 cm, midway between the lower border of the ribs and the iliac crest at the widest portion, over light clothing, using a soft measuring tape, without any pressure to the body.
For measurements of systolic (SBP) and diastolic blood pressure (DBP), after a 15-min rest in upright position, two measurements of blood pressure were taken on the right arm, during a standardized mercury sphygmomanometer; the mean of the two measurements was considered as the participant's blood pressure.

Demographic, Anthropometric and Clinical Measures
Trained interviewers collected information using standard questionnaires. Detailed measurements of variables in TLGS have been reported elsewhere [32]. Smoking status was obtained using face-to-face interviews; subjects who smoked daily or occasionally were considered current smokers. Weight was measured to the nearest 100 g using digital scales, while the subjects were minimally clothed, without shoes. Height was measured to the nearest 0.5 cm, in a standing position without shoes, using a tape meter. Body mass index (BMI) was calculated as weight (kg) divided by square of the height (m 2 ). Waist circumference (WC) was measured to the nearest 0.1 cm, midway between the lower border of the ribs and the iliac crest at the widest portion, over light clothing, using a soft measuring tape, without any pressure to the body.
For measurements of systolic (SBP) and diastolic blood pressure (DBP), after a 15-min rest in upright position, two measurements of blood pressure were taken on the right arm, during a standardized mercury sphygmomanometer; the mean of the two measurements was considered as the participant's blood pressure.

Biochemical Measures
Fasting blood samples were taken after 12-14 h, from all study participants at baseline and at follow-up phases. Serum creatinine levels were assayed using kinetic colorimetric Jaffe method. Fasting plasma glucose (FPG) was measured by the enzymatic colorimetric method using glucose oxidase. The standard 2-h post-challenge plasma glucose (2 h-PCPG) test was performed using oral administration of 82.5 g glucose monohydrate solution (equivalent to 75 g anhydrous glucose) for all individuals who were not on glucose lowering drugs.
Triglyceride (TG) level was measured by enzymatic colorimetric analysis with glycerol phosphate oxidase. High-density lipoprotein cholesterol (HDL-C) was measured after precipitation of the apolipoprotein B containing lipoproteins with phosphotungstic acid. Analyses were performed using Pars Azmoon kits (Pars Azmoon Inc., Tehran, Iran) and a Selectra 2 auto-analyzer (Vital Scientific, Spankeren, The Netherlands). Both inter-and intra-assay coefficients of variation of all assays were <5%.
To develop a validation study for dietary NO 3 − and NO 2 − , urine NO 3 − and NO 2 − concentration was measured in a sub-sample of population (n = 251), by a rapid, simple spectrophotometric method [34][35][36].

Dietary Assessment
A validated 168-item food frequency questionnaire (FFQ) was used to assess typical food intakes over the previous year. Trained dietitians, with at least 5 years of experience in the TLGS survey, asked participants to designate their intake frequency for each food item consumed during the past year on a daily, weekly, or monthly basis. Portion sizes of consumed foods reported in household measures were then converted to grams [33]. The validity of the food frequency questionnaire has been previously evaluated by comparing food groups and nutrient values determined from the questionnaire with values estimated from the average of twelve 24-h dietary recall surveys and the reliability has been assessed by comparing energy and nutrient intakes from two FFQ; Pearson correlation coefficients and intra-class correlation for energy and nutrients showed acceptable agreements between FFQ and twelve 24-h dietary recall surveys, and FFQ1 and FFQ2 [37].
Since the Iranian Food Composition Table is incomplete, and has limited data on nutrient content of raw foods and beverages, to analyze foods and beverages for their energy and nutrient content (except NO 3 − andNO 2 − ), the US Department of Agriculture Food Composition Table was used [38].

Definition of Terms and Outcomes
The HTN was defined as SBP ≥ 140 or mmHg DBP ≥ 90 mmHg, or self-reported taking blood pressure lowering medication [40].
Incident CKD was defined as estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m 2 occurring at any time during the follow-up period; this corresponds to stage 3 to stage 5 CKD based on the Kidney Disease Outcomes and Quality Initiative guidelines [41]. To calculate eGFR, the CKD Epidemiology Collaboration (EPI) equation was used. As a single equation CKD-EPI has been expressed as follows: In this equation, S cr is serum Cr in mg/dL; κ is 0.7 and 0.9 for men and women, respectively, α is −0.329 and −0.411 for men and women, respectively; min indicates the minimum of S cr /κ or 1, and max indicates maximum of S cr /κ or 1 [42].
The family history of premature cardiovascular disease was obtained by asking participants whether any member in their first-degree relatives had experienced a fatal or non-fatal myocardial infarction, stroke, or sudden cardiac arrest; the event was considered premature if it occurred in persons <55 years of age in male relatives and <65 years of age in female relative [43]. Type 2 diabetes (T2D) was defined as FPG ≥ 7 mmol/L or 2 h-PCPG ≥ 11.1 mmol/L, or taking antidiabetic medication [44].

Statistical Analyses
Dietary intakes of NO 3 − and NO 2 − and other nutrients were adjusted for total energy intake, according to residuals methods [45]. The incidence of HTN and CKD over the follow-up period was considered as a dichotomous variable (yes/no) in the models. The mean and standard deviation (SD) values, and the frequency (%) of baseline characteristics of the participants with and without HTN and CKD were compared using independent t test or chi square test, respectively. A univariate analysis was performed to identify potential covariates and the variables with P E < 0.2 in the univariate analyses were selected for the final multivariable models. Potential confounding variables adjusted in the final regression model were included baseline SBP (mmHg), baseline DBP (mmHg), WC (cm), family history of premature cardiovascular disease (yes/no), smoking (yes/no), lipid-lowering drugs (yes/no), aspirin (yes/no), dietary intakes of fiber (g/day), fat (g/day), potassium (mg/day) and sodium (mg/day) for HTN [43], and age (years), sex (male/female), type 2 diabetes (yes/no), HTN (yes/no), eGFR (mL/min/1.73 m 2 ), smoking (yes/no), dietary intakes of fat (g/day), protein (g/day), potassium (mg/day) and sodium (mg/day) for CKD [46]. The association between different intake levels of NO 3 − and NO 2 − intake with incident HTN and CKD was assessed by multivariate adjusted odds ratios (ORs) with 95% confidence interval (CI) using binary logistic regression analysis. For risk covariates with more than 2 categories, the first category was considered as the reference group, in the model. To assess the overall trends of odds ratios, the median of each tertile was used as a continuous variable in logistic regression models. All statistical analyses were conducted using SPSS (Version 16.0, IBM; Chicago, IL, USA), and p values < 0.05 were considered significant.
In our population, the major contributors to NO 3 − intakes were vegetables (46.1%) and grains (28.8%). Baseline characteristics of the participants for incident CKD are shown in Supplementary Materials Table S3. Mean (SD) age of the study participants was 33.9 (15.4) years and 40.8% were men. Mean (SD) BMI was 27.4 (4.8) kg/m 2 , at baseline. Over a median 5.8 years of follow-up, a total of 306 cases of CKD were diagnosed (cumulative incidence rate = 17.2%). The CKD patients had higher BMI, WC, blood pressures, FPG, TG to HDL-C ratio, and lower eGFR rate, at baseline (p for all < 0.05). Compared to CKD patients, non-CKD subjects had higher intake of NO 3 − (467 vs. 443 mg/day, p = 0.02) at baseline, whereas no significant difference was observed in NO 2 − intake between the groups. Association between NO 3 − and NO 2 − intake and the risk of HTN after 5.8 years of follow-up are shown in Table 3. We did not observe any significant association between intake of NO 3 − and the risk of HTN in the logistic regression models. Compared to the lowest tertile category (median intake < 6.04 mg/day), the highest intake (median intake ≥ 12.7 mg/day) of dietary NO 2 − was accompanied with a significant reduced risk of HTN, in the fully adjusted model (OR = 0.58, 95% CI = 0.33-0.98; p for trend = 0.054). The incidence of CKD across tertile categories of NO 3 − and NO 2 − intake are shown in Table 4.
After adjustment of major potential confounding variables, dietary intake of NO 3 − had no significant association with the risk of CKD whereas highest compared to the lowest tertile of dietary NO 2 − was accompanied with a reduced risk of CKD (OR = 0.50, 95% CI = 0.24-0.89, p for trend = 0.07). Odds ratio (95% CI); logistic regression models were used. The first tertile of NO 3 − (<359 mg/day) and NO 2 − intake (<7.58 mg/day) was considered as reference group. Model 1: Adjusted for age (years), sex (male/female), systolic and diastolic blood pressure (mmHg), waist circumference (cm), family history of premature cardiovascular disease (yes/no), and smoking (yes/no), lipid-lowering drugs (yes/no), aspirin (yes/no); Model 2: Additional adjustment for dietary intake of total fiber (g/day), fat (g/day), potassium (mg/day), and sodium (mg/day). Median intake of dietary NO 3 − was 288, 428, and 613 mg/day, in the first, second, and third tertile categories. Median intake of dietary NO 2 − was 6.04, 9.00, and 12.7 mg/day, in the first, second, and third tertile categories. HTN: Hypertension. Odds ratio (95% CI); logistic regression models were used; the first tertile of NO 3 − (<365 mg/day) and NO 2 − intake (<7.69 mg/day) was considered as reference group; the number of case/total was 116/593, 103/594, and 99/593 in the first, second, and third tertile categories of dietary nitrate intakes. The number of case/total was 110/593, 107/594, and 101/593 in the first, second, and third tertile categories of dietary nitrite intakes; Model 1: Adjusted for age (years), sex (male/female), diabetes (yes/no), hypertension (yes/no), eGFR (mL/min/1.73 m 2 ), and smoking (yes/no); Model 2: Additional adjustment for dietary intake protein (g/day), fat (g/day), potassium (mg/day), and sodium (mg/day); Median intake of dietary NO 3 − was 291, 431, and 619 mg/day, in the first, second, and third tertile categories; Median intake of dietary NO 2 − was 6.14, 9.08, and 12.8 mg/day, in the first, second, and third tertile categories.

Discussion
In this longitudinal study, we investigated the potential impact of habitual dietary NO 3 − and NO 2 − intake on the risk of HTN and CKD, in the framework of a population-based study, for the first time. Higher dietary NO 2 − intake was significantly associated with a reduced risk of HTN and CKD, independent of the major potential risk factors. Compared to CKD patients, non-CKD subjects had higher intake of NO 3 − (467 vs. 443 mg/day, p = 0.02) at baseline; however, dietary NO 3 − intake was not related to incidence of either HTN of CKD after a median 5.8 years of follow-up. Most recent findings imply beneficial cardio-renal protective and antihypertensive outcomes following short-term administration of inorganic NO 3 − [16,19,24,47]. The underlying mechanisms for the favorable effects of inorganic NO 3 − and NO 2 − in human subjects are still not fully understood, but it has been proposed that NO 2 − could be a stable endocrine carrier and transducer of NO-like bioactivity within the circulation; systemic vasodilatation through the NO-cGMP pathway has been suggested as the acute effects of dietary NO 3 − and NO 2 − [28,29]. The novel mechanisms recently investigated in a model of natural aging-related cardiovascular and metabolic abnormalities, suggest that inorganic NO 3 − mediates its therapeutic effects through restored cGMP signaling and increased NO bioavailability, decreased ANG II type 1 receptor expression, improved endothelial function, increased insulin release and reduced NADPH oxidase activity and superoxide generation [25]. Beneficial effects of NO 3 − and NO 2 − on renal function may be explained by promoting the NO 3 − -NO 2 − -NO pathway, attenuation of ANG II-induced hypertension, and reducing constriction of renal afferent arterioles [48,49]. It also has been shown that NO 3 − supplementation could normalize elevated plasma creatinine levels and improve glomerular function during aging [25] and prevent renal dysfunction in experimental models of compromised kidney function and cardiovascular disease [24]. In addition, experimental studies have indicated that inorganic nitrite may protect from kidney injuries following acute ischemia-reperfusion [50,51] We could not estimate NO 3 − /NO 2 − exposure from drinking water due to lack of data for drinking water NO 3 − /NO 2 − contents and individuals' information regarding water intake, at baseline.
However, previous studies showed that NO 3 − /NO 2 − concentration of drinking water was lower than the standard limits (50 mg/L) [58]; considering the low amount of water intake among the Iranian population (~0.96 L) [59], it seems that NO 3 − /NO 2 − intakes from drinking water are relatively low.
Furthermore, our recent study in district 13 of Tehran showed that NO 3 − and NO 2 − levels of drinking water was 32.8 ± 9.9 and 2.6 ± 0.5 mg/L, respectively, and estimation of NO 3 − /NO 2 − intakes from drinking water in a subsample of TLGS population showed a relatively low contribution of drinking water in overall NO 3 − /NO 2 − exposure compared to its dietary sources (6.7% and 26.6% for NO 3 − and NO 2 − , respectively).
Estimates of NO 2 − from meat are likely to be inaccurate as most of the NO 2 − forms nitrosylmyoglobin. Furthermore, due to potential changes in an individual's diet and NO 3 − /NO 2 − content of food items, as well as changes in other risk factors of HTN and CKD over the time of study follow-up, some degree of misclassification might have occurred which could lead to biased estimated hazard ratios towards the null, as inherent to any prospective study.
In conclusion, this prospective study suggests that a higher intake of dietary NO 2 − can decrease the risk of developing both HTN and CKD. Although higher intake of NO 3 − was associated with lower incidence of CKD at baseline, we did not find that differences in NO 3 − intake influenced incidence of HTN or CKD during the study period of 6 years. Our findings, especially in the case of NO 2 − supplementation, support previous experimental and clinical studies that suggest the therapeutic value of boosting the NO 3 − -NO 2 − -NO pathway.

Perspectives
In this study, dietary NO 2 − intake had a protective effect against CKD and HTN, and both of them are associated with decreased nitric oxide availability. Dietary NO 3 − and NO 2 − could act as precursors for nitric oxide production in case of its deficiency. It seems that intake of NO 3 − /NO 2 − should be taken into consideration in dietary assessments, in particular in patients with CKD and HTN. In addition, since NO 3 − /NO 2 − therapy could easily be achieved through nutrition-based interventions, it could be speculated that such intervention contributes to the future management of HTN and kidney diseases.