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
]. 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
]. 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
In recent years, following the discovery of potential ability of inorganic nitrate (NO3−
) and nitrite (NO2−
) 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 NO3−
] shifted towards therapeutic properties of these compounds in cardiometabolic disorders [11
]. Theoretically, reductions of NO3−
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 NO3−
have been investigated as potential therapeutic options in cardiovascular disease, including HTN, and in states renal dysfunction [23
]. Currently, a large body of evidence supports a crucial role of NO3−
in the regulation and modulation of blood flow, endothelial function, and blood pressure [26
]. Pre-clinical studies also confirm protective effects of NO3−
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 NO3−
-containing vegetables against development of HTN, we speculated that the observed effect may be related to NO3−
]; after development of a valid database of NO3−
content of food items [31
], we expanded our hypothesis in the framework of the current study to clarify potential effects of NO3−
on the risk of HTN and CKD.
To the best of our knowledge, the potential impact of dietary NO3− and NO2− 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 NO3− and NO2−. The main focus in this study, therefore, was to ascertain whether regular intake of NO3− and NO2− could predict the occurrence of HTN and chronic kidney disease (CKD) among an Iranian population, during a 6-year follow-up.
Mean (SD) intakes of dietary NO3− and NO2− was 455 (188) and 9.4 (3.6) mg/day, respectively. In our population, the major contributors to NO3− intakes were vegetables (46.1%) and grains (28.8%). Dietary intakes of NO2− from animal sources accounted for 42.4% of daily mean intake of NO2− and the remainder of NO2− intake was derived from plant sources. The major contributors to NO2− intake were white rice (17.1%), chicken meat (11.7%), yogurt (6.6%), tomato (5.3%), sausages (4.7%), lamb meat (3.5%), cucumber (3.3%).
Baseline characteristics and dietary intakes of the participants are compared across tertile categories of dietary intakes of NO3−
in HTN-free subjects, in Table 1
and Table 2
, respectively. Participants in the highest compared to the lowest tertile of NO3−
, were less likely to be smoker (8.9 vs. 13.1, p
< 0.05); there was no significant difference in lipid lowering drug and aspirin intakes, anthropometric measures, systolic and diastolic blood pressures, FPG and TG to HDL-C ratio across NO3−
tertiles. All components of the diet had increasing trend across increasing intakes of NO3−
. There was no significant difference in the rate of incident-case of HTN across NO3−
tertiles, after 5.8 years of follow-up. Baseline characteristics of the participants are compared across tertile categories of dietary intakes of NO3−
in CKD-free subjects, in Supplementary Materials Table S1
. Participants in the highest compared to the lowest tertile of NO3−
, were more likely to be older (36.7 vs. 30.6 years, p
< 0.05), and had lower serum creatinine levels (90.8 vs. 93.2 µmol/L, p
< 0.05); there was a non-significant lower rate of incident-CKD in the highest compared to the lowest tertile of dietary NO3−
(16.7% vs. 19.6%) and NO2−
(17.0% vs. 18.5%) intakes.
Baseline characteristics of the study participants for incident HTN according to outcome status are shown in Supplementary Materials Table S2
. Mean (SD) age of the study participants was 36.6 (12.4) years and 42.9% were men. Mean (SD) BMI was 26.3 (4.7) kg/m2
, at baseline. Overall, 291 new cases HTN were identified after a median follow-up of 5.8 years; the corresponding cumulative incidence was 15.5%. Compare with non-HTNs, hypertensive subjects were more likely to be older, and had higher BMI, WC, blood pressures, FPG, TG to HDL-C ratio, prevalent T2D, creatinine levels and lower eGFR (p
for all < 0.05). Mean (SD) baseline intake of NO3−
was 455 ± 188 and 9.47 ± 3.61 mg/day, and there was no difference in dietary intakes of NO3−
between the groups.
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/m2
, 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 NO3−
(467 vs. 443 mg/day, p
= 0.02) at baseline, whereas no significant difference was observed in NO2−
intake between the groups.
Association between NO3−
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 NO3−
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 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 incidence of CKD across tertile categories of NO3−
intake are shown in Table 4
. After adjustment of major potential confounding variables, dietary intake of NO3−
had no significant association with the risk of CKD whereas highest compared to the lowest tertile of dietary NO2−
was accompanied with a reduced risk of CKD (OR = 0.50, 95% CI = 0.24–0.89, p
for trend = 0.07).
In this longitudinal study, we investigated the potential impact of habitual dietary NO3− and NO2− intake on the risk of HTN and CKD, in the framework of a population-based study, for the first time. Higher dietary NO2− 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 NO3− (467 vs. 443 mg/day, p = 0.02) at baseline; however, dietary NO3− 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 NO3−
]. The underlying mechanisms for the favorable effects of inorganic NO3−
in human subjects are still not fully understood, but it has been proposed that NO2−
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 NO3−
]. The novel mechanisms recently investigated in a model of natural aging-related cardiovascular and metabolic abnormalities, suggest that inorganic NO3−
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 NO3−
on renal function may be explained by promoting the NO3−
–NO pathway, attenuation of ANG II-induced hypertension, and reducing constriction of renal afferent arterioles [48
]. It also has been shown that NO3−
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
]. Hence, the above mentioned mechanisms might justify 42% and 50% decreased risk of HTN and CKD in relation to dietary intakes of NO2−
more than ~10 mg/day, in our study population.
In contrast to the vast majority of experimental findings indicating renoprotective properties of NO3−
, there have been some concerns regarding its harmful effects for humans, especially when used in high doses to improve exercise performance. To address this challenging issue, a recent clinical study investigated the effects of potassium NO3−
(450 mg/day) on GFR, and urine output for creatinine, albumin, and urea, in young male during a cycling exercise condition. This study reported no adverse effects on renal function, over one week period of NO3−
]. Currently, there are no further clinical studies to confirm or reject beneficial effects of inorganic NO3−
on kidney function.
An overview of the current literature displays lack of epidemiological evidence regarding cardiorenal outcomes of NO3−
in the context of daily dietary intake. The only relevant studies in this case, was our previous cohort with a 3-year follow-up that showed a protective effect against HTN and no significant impact on CKD following higher consumption of NO3−
-containing vegetables [30
]. Lack of information regarding true NO3−
content of the vegetables was an important limitation of these works; we also did not observe any difference between categories of NO3−
-containing vegetables (including low-, medium- and high-NO3−
) in relation to HTN, so we concluded that other bioactive compounds, including phytochemicals and antioxidant components, may be involved in the hypotensive effect of these vegetables. In the current study, NO3−
from vegetable sources was not related to risk of HTN (OR = 0.97, 95% CI = 0.67–1.42, and OR = 0.98, 95% CI = 0.63–1.52, in the second and third quartile categories, respectively) and CKD (OR = 1.31, 95% CI = 0.88–1.93, and OR = 0.93, 95% CI = 0.57–1.50, in the second and third quartile categories, respectively).
The usual dietary consumption of NO3−
in our study was higher than in other previous reports such as the Shanghai Women’s Health Study, National Institutes of Health/American Association of Retired Persons (NIH-AAPR) diet and health study, which estimated dietary intakes of ~300 and 100 mg/day for NO3−
and 1.4 and 1.0 mg/day for NO2−
]. Moreover, our intakes was approximately twice the acceptable daily intake (ADI) values, defined as 3.7 and 0.06 mg/kg body weight for NO3−
, respectively [56
]. Major sources of NO3−
intakes were grains and vegetables; due to a relatively high NO3−
concentration in our traditional and industrial breads (50.0 mg·100 g−1
], and high proportion of breads (320 g/day) in the dietary pattern of the Iranian population [57
exposure from this food group was considerable in our population. In our previous study we indicated that mean NO3−
levels in 68.3% of lettuce, 92.5% of potato, 90.9% of radish, and 51.0% of cabbage samples exceeded the maximum limits legislated by European countries for trade of vegetables; moreover, mean NO2−
contents of fruit samples were also relatively high [31
]. High intake of NO3−
in our population, therefore, may be attributed to either high content of NO3−
in Iranian foods or high intake of NO3−
-containing foods. Hence, considering the fact that most dietary substances are rather low in NO2−
, and that the vast majority of NO2−
is likely to be derived from reduction of dietary NO3−
], rather than dietary NO2−
per se, the correlation between dietary NO2−
and its urinary values, we observed in a validity study, was rather week.
Our study had some strengths and limitations. The large, prospective population-based design, a high rate of follow-up completeness, and use of a validated comprehensive FFQ to assess regular dietary intakes of the participants provided us an opportunity to examine the potential effect of NO3−
on the risk of HTN and CKD, relationship that have not been previously reported. Estimation of NO3−
based on measured values in frequently consumed food items among our population [31
], compared to other previous cohorts which have relied on historic literature values, may fully reflect the accurate NO3−
exposure from the diet.
We could not estimate NO3−
exposure from drinking water due to lack of data for drinking water NO3−
contents and individuals’ information regarding water intake, at baseline. However, previous studies showed that NO3−
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 NO3−
intakes from drinking water are relatively low. Furthermore, our recent study in district 13 of Tehran showed that NO3−
levels of drinking water was 32.8 ± 9.9 and 2.6 ± 0.5 mg/L, respectively, and estimation of NO3−
intakes from drinking water in a subsample of TLGS population showed a relatively low contribution of drinking water in overall NO3−
exposure compared to its dietary sources (6.7% and 26.6% for NO3−
Estimates of NO2− from meat are likely to be inaccurate as most of the NO2− forms nitrosylmyoglobin. Furthermore, due to potential changes in an individual’s diet and NO3−/NO2− 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 NO2− can decrease the risk of developing both HTN and CKD. Although higher intake of NO3− was associated with lower incidence of CKD at baseline, we did not find that differences in NO3− intake influenced incidence of HTN or CKD during the study period of 6 years. Our findings, especially in the case of NO2− supplementation, support previous experimental and clinical studies that suggest the therapeutic value of boosting the NO3−–NO2−–NO pathway.