SDMA | Microrna-2

Urinary albumin-to-creatinine ratio is inversely related to nitric oxide synthesis in young black adults: the African-PREDICT study

Ashleigh Craig1 ● Catharina M. C. Mels1,2 ● Aletta E. Schutte1,2,3 ● Alexander Bollenbach4 ● Dimitrios Tsikas4 ●
Edzard Schwedhelm5,6 ● Ruan Kruger1,2

Received: 4 May 2020 / Accepted: 21 June 2020
© The Japanese Society of Hypertension 2020

Abstract
Hypertension is common in black populations and is known to be associated with low nitric oxide (NO) bioavailability. We compared plasma and urinary NO-related markers and plasma creatine kinase (CK) levels between young healthy black and white adults along with the associations of these markers with the urinary albumin-to-creatinine ratio (uACR), which is a surrogate marker of endothelial and kidney function. We included 1105 participants (20–30 years). We measured the uACR,plasma CK, plasma and urinary arginine, homoarginine, asymmetric (ADMA) and symmetric dimethylarginine (SDMA),
urinary ornithine/citrulline, nitrate and nitrite, and malondialdehyde (MDA). In addition, the urinary nitrate-to-nitrite ratio (UNOxR) was calculated and used as a measure of circulating NO bioavailability. The uACR was comparable between the groups, yet the black group had lower urinary nitrate (by −15%) and UNOxR values (by −18%) (both p ≤ 0.001), higher plasma (by +9.6%) and urinary (by +5.9%) arginine (both p ≤ 0.004), higher plasma (by +13%) and urinary (by +3.7%) ADMA (both p ≤ 0.033), and higher CK (by +9.5%) and MDA (by +19%) (both p < 0.001) compared with white adults. Plasma and urinary homoarginine were similar between the groups. In the multiple regression analysis, we conﬁrmed the inverse associations of the uACR with both plasma (adj. R2 = 0.066; β = −0.209; p = 0.005) and urinary (adj. R2 = 0.066; β = −0.149; p = 0.010) homoarginine and with the UNOxR (adj. R2 = 0.060; β = −0.122; p = 0.031) in the black group only. The overall less favorable NO proﬁle and higher CK and MDA levels in the black cohort along with the adverse associations with the uACR may reﬂect the vulnerability of this cohort to the early development of hypertension.
Keywords Urinary albumin-to-creatinine ratio ● Nitric oxide ● Homoarginine ● Urinary nitrate-to-nitrite ratio ● Hypertension

Introduction
Supplementary information The online version of this article (https://

doi.org/10.1038/s41440-020-0514-1) contains supplementary material, which is available to authorized users.

* Ruan Kruger [email protected]

1 Hypertension in Africa Research Team (HART), North-West University, Potchefstroom, South Africa
2 MRC Research Unit for Hypertension and Cardiovascular Disease, North-West University, Potchefstroom, South Africa
3 School of Public Health and Community Medicine, University of New South Wales and The George Institute for Global Health, Sydney, Australia
4 Institute of Toxicology, Core Unit Proteomics, Hannover Medical School, Hannover, Germany
5 Institute of Clinical Pharmacology and Toxicology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
6 Deutsches Zentrum fuer Herz-Kreislauf-Forschung E.V. (DZHK), Partner Site Hamburg/Kiel/Lüebeck, Hamburg, Germany
Increased blood pressure is a main contributing factor to cardiovascular outcomes [1], yet the origins of the early phases of disease development remain unclear. Noticeably, a cardiovascular risk factor such as hypertension—which is
common in black populations [2]—may be partly due tounderlying endothelial dysfunction [3, 4]. Endothelial dys-
function is measured by several noninvasive methods, including ﬂow-mediated dilation, which has been widely used in the last few decades [5, 6]. However, more recently, the urinary albumin-to-creatinine ratio (uACR) has been introduced as a surrogate marker of both vascular endo-
thelial and renal function [7–9]. Moreover, an elevated uACR has also been recognized to predict hypertension [10]
and mortality [11] in the general population.

One of the important mediators of endothelial function is nitric oxide (NO). NO is synthesized from L-arginine (Arg) and L-homoarginine (hArg), which serve as substrates in areaction catalyzed by NO synthase (NOS) [12]. Alter- natively, NO can be formed via the reduction of the inor- ganic anions nitrate and nitrite, which are the main metabolites of NO [13]. Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are inhibitors of NOS activity and Arg transport [14]. Impair- ment of the synthesis or bioavailability of NO results in a proconstrictive phenotype [15] and has been reported to be more common in black populations [16] and hypertensive patients [10]. hArg is synthesized by L-Arg:glycine amidi- notransferase (AGAT) [17], an enzyme that is also responsible for the synthesis of the energy-related metabo- lite creatine [18]. Creatine is the substrate of creatine kinase (CK), and its synthesis requires larger quantities of available L-Arg than NO synthesis [18, 19]. This may suggest that the CK system may be sensitive to a reduction in Arg bioavailability. NO synthesis from Arg is therefore regu- lated not only by Arg availability but also by competition for substrates between NO and creatine synthesis.

Studies comparing the uACR and CK levels in black and white populations have provided consistent ﬁndings of
increased uACR [20–22] and CK levels [23, 24] in black populations. However, reports on NO proﬁles in black and
white populations have been inconsistent [20, 25, 26]. One study reported a greater NO synthesis capacity in a black population with higher levels of Arg [20], while another study observed reduced NO production and lower levels of Arg in black men [25]. In addition, a study presenting the ﬁrst evaluation of the urinary nitrate-to-nitrite ratio (UNOxR) suggested that the UNOxR was a better measure of carbonic acid anhydrase-dependent nitrite reabsorption and reported a lower UNOxR in black boys compared with that in age- matched white boys, potentially suggesting an association of reduced NO synthesis with black ethnicity [27]. There have also been inconsistent ﬁndings for the endogenous NOS inhibitor ADMA, which is considered a marker of endothelial dysfunction in adults [14, 28]. Circulating ADMA was found to be lower in black populations [20, 29], higher in black Africans [26] or similar [25] when compared with that in white individuals. ADMA and SDMA are produced by the catalytic dimethylation by protein-Arg methyltransferase (PRMT) of proteinic Arg residues. ADMA and SDMA circulate in the blood and are excreted in the urine, and ADMA is also metabolized by dimethylarginine dimethylaminohydrolase (DDAH) [30].

Due to the varying ﬁndings for the NO proﬁle in black and white populations, a deeper look into NO proﬁles among these populations is warranted. Furthermore, no prior studies have examined both the plasma and urinary NO proﬁles of young and apparently healthy individuals in bi-ethnic populations. Thus, in young black and white adults, we compared NO-related markers derived from urine and plasma along with the respective plasma CK levels anddetermined their relationship with the uACR as a measure of endothelial and kidney function.

Methodology

The African Prospective Study on the Early Detection and Identiﬁcation of Cardiovascular Disease and Hypertension (African-PREDICT) screened and assessed 1202 apparently healthy volunteers (aged 20–30 years). In this cross-
sectional study, we included data from 1105 participants
who were stratiﬁed according ethnicity, i.e., black (n = 539) and white (n = 566) men and women, after the exclusion of participants with nitrite outliers and those with missing NO- related and uACR data (n = 97).
The population and the protocol for the African- PREDICT study have been described elsewhere [31]. Brieﬂy, participants with an ofﬁce blood pressure >140/90 mmHg during screening or with any self-reported diseases or risk factors that may inﬂuence cardiovascular health, an internal ear temperature >37.5 °C, human immunodeﬁ- ciency virus, diabetes mellitus, liver disease, cancer, tuberculosis or renal disease, as well as the use of chronic medication were excluded from the African-PREDICT study. Pregnant and lactating women were also excluded due to the known inﬂuences of hormones on cardiovascular health.

Participants were fully informed about the objectives of the study, and written informed consent was acquired from each participant. The African-PREDICT study was con- ducted according to the ethical principles of the Declaration of Helsinki [32] and was approved by the Health Research Ethics Committee of North-West University.

Anthropometric measures

All anthropometric procedures were performed according to speciﬁc guidelines set out by the International Society for the Advancement of Kinanthropometry [31, 33]. The waist circumference (cm) was obtained using a standard protocol (Lufkin Steel Anthropometric Tape; W606PM; Lufkin; Apex; USA). The body mass index (BMI) (weight (kg)/ square height (m2)) (SECA portable 213 stadiometer; SECA 813 electronic scale; Hamburg, Germany) and waist-to-hip ratio (waist circumference (cm)/hip circumference (cm)) of each participant were then calculated.

Cardiovascular measuresWith a Dinamap® ProCare 100 Vital Signs Monitor, the ofﬁce blood pressure was measured in the left arm and then in the right arm in duplicate, which was followed by a repeated measurement in the left upper arm (GE Medical

Table 1 General characteristics of young adults stratiﬁed according to ethnicity

Age (years) Black (n = 539)

24.4 ± 3.14 White (n = 566)

24.6 ± 3.05 p value

0.41
Sex men (n %) 261 (48%) 271 (47%) 0.86
Body composition
Waist circumference (cm) 77.6 ± 11.1 82.7 ± 13.7 <0.001
Body height (cm) 164.45 ± 8.48 172.49 ± 8.73 <0.001
Body weight (kg) 71.24 ± 17.97 72.20 ± 17.06 0.36
Body mass index (kg/m²) 24.6 ± 5.77 25.6 ± 5.42 0.002
Cardiovascular measures
Systolic blood pressure (mmHg) 118 ± 12 117 ± 12 0.052
Diastolic blood pressure (mmHg) 80 ± 9 78 ± 8 <0.001
Central systolic blood pressure (mmHg) 110 ± 9 106 ± 9 <0.001
Biochemical measures
Urinary albumin-to-creatinine ratio 0.48 (0.15–2.00) 0.47 (0.17–2.01) 0.34
Total cholesterol (mmol/L) 3.73 (2.68–5.40) 4.61 (3.19–6.50) <0.001
High-density lipoproteins (mmol/L) 1.26 ± 0.336 1.36 ± 0.416 <0.001
Low-density lipoproteins (mmol/L) 2.33 (1.30–3.98) 2.98 (1.80–4.81) <0.001
Triglycerides (mmol/L) 0.72 (0.37–1.41) 0.98 (0.44–2.36) <0.001
C-reactive protein (mg/L) 1.27 (0.13–12.9) 0.99 (0.10–10.3) 0.014
eGFR (mL/min/1.73 m²) 118 ± 14.9 106 ± 16.0 <0.001
Glucose (mmol/L) 4.35 (2.95–5.56) 4.70 (3.71–5.64) 0.001
Glycated hemoglobin (%) 5.44 ± 0.30 5.20 ± 0.28 <0.001
Malondialdehyde (μM/mM creatinine) 0.16 (0.07–0.51) 0.13 (0.06–0.32) <0.001
Gamma-glutamyl transferase (U/L) 24.9 (10.4–75.9) 18.7 (7.84–51.0) <0.001
Cotinine (ng/mL) 3.77 (1.00–343) 3.25 (1.00–313) 0.31
Creatine kinase (ng/mL)a 1.59 (0.64–4.58) 1.35 (0.57–3.80) <0.001
Lifestyle risk factors
Smoke (n %) 154 (29%) 131 (23%) 0.040
Alcohol (n %) 146 (27%) 160 (28%) 0.35
Values are arithmetic mean ± standard deviation or geometric mean (5th and 95th percentiles) for logarithmically transformed variables. Bold values denote statistical signiﬁcance (p< 0.05)
n number of participants, eGFR estimated glomerular ﬁltration rate, hba1c glycated hemoglobin
aCreatine kinase was adjusted for age, sex, and energy expenditureSystems, Milwaukee, USA). In this study, the mean left blood pressure measurement was used in the analyses. The systolic blood pressure and diastolic blood pressure were determined from each measurement. With a SphygmoCor XCEL device (AtCor Medical Pty. Ltd, Sydney, New South Wales, Australia), supine central systolic blood pressure readings were derived using pulse wave analysis [34].

Biochemical analyses

Participants were required to fast for at least 8 h before they provided an early morning spot urine sample. Blood sam- ples were obtained by a registered research nurse at the Hypertension Clinic of North-West University.
Basic serum analyses included lipids (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglycerides), gamma-glutamyl transferase,
creatinine, and high-sensitivity C-reactive protein (Cobas Integra 400 plus Roche, Basel Switzerland). Serum cotinine levels were determined with a chemiluminescence method with an Immulite system (Siemens, Erlangen, Germany). CK was determined with the electrochemiluminescence method by using an E411 (Roche, Basel Switzerland). Sodium ﬂuoride plasma glucose (Siemens, Erlangen, Ger- many) and EDTA whole blood glycated hemoglobin were also determined (Cobas Integra 400 plus Roche, Basel Switzerland). The intra-assay variability and interassay variability of all variables were below 10%. Urinary albu- min (mg/L) and creatinine (mmol/L) were determined (Cobras Integra® 400 plus, Roche, Basel, Switzerland), and the uACR was calculated. Furthermore, the Chronic Kidney Disease Epidemiology formula was utilized to calculate the estimated glomerular ﬁltration rate (eGFR) from the serum creatinine values [35].

Table 2 Plasma and urinary nitric oxide-related markers of the study population stratiﬁed according to ethnicity

Black (n = 539) White (n = 566) p value
Plasma nitric oxide-related markers†
Arginine (μM)
92.7 (60.3–149)
83.8 (56.7–117)
<0.001
Homoarginine (μM) 2.05 (0.97–4.33) 2.05 (1.06–3.77) 0.99
ADMA (μM) 0.60 (0.39–0.92) 0.52 (0.31–0.78) <0.001
SDMA (μM) 0.533 ± 0.142 0.562 ± 0.129 0.012
Urinary nitric oxide-related markers
Arginine (μM/mM creatinine) 2.37 (1.38–4.18) 2.23 (1.31–3.95) 0.004
Homoarginine (μM/mM creatinine) 0.15 (0.04–0.52) 0.15 (0.05–0.63) 0.81
ADMA (μM/mM creatinine) 3.82 (2.27–6.01) 3.68 (2.15–5.94) 0.033
SDMA (μM/mM creatinine) 3.73 (2.44–5.36) 3.89 (2.59–5.76) 0.022
Ornithine/citrulline (μM/mM creatinine) 3.48 (2.19–5.49) 3.57 (1.91–5.51) 0.017
Nitrate (μM/mM creatinine) 59.6 (32.7–127) 70.5 (37.8–146) <0.001
Nitrite (μM/mM creatinine) 0.29 (0.10–0.90) 0.28 (0.11–0.95) 0.51
Urinary nitrate-to-nitrite ratio 204 (64.49–573) 248 (88.19–601) <0.001
Values are arithmetic mean ± standard deviation or geometric mean (5th and 95th percentiles) for logarithmically transformed variables. Bold values denote statistical signiﬁcance (p <0.05)
n number of participants
†Samples measured in plasma (black n = 279; white n = 282)

The plasma NO-related markers (Arg, hArg, ADMA, and SDMA) of the ﬁrst 561 participants were quantiﬁed by liquid chromatography-tandem mass spectrometry [36, 37]. Urinary NO-related markers (Arg, hArg, ADMA, SDMA, ornithine/citrulline, nitrite, and nitrate) as well as mal- ondialdehyde (MDA), which is a biomarker of oxidative stress, and creatinine were measured simultaneously by
gas chromatography–mass spectrometry [38]. All urinary NO-related data were normalized based on creatinine
excretion and are expressed as μM analyte to mM creatinine (μM/mM). The UNOxR values were calculated by dividing the urinary nitrate concentration by the urinary nitrite con- centration [27].

Statistical analysis

For the statistical analyses, IBM®, SPSS® version 26 (IBM Corporation, Armonk, New York) was used. We tested the interactions of ethnicity with the association with the uACR with both plasma and urinary NO-related markers. Vari-
ables were tested for normality using the Kolmogorov–Smirnov test and QQ plots. Non-Gaussian variables were log10-transformed. Data were expressed as
the mean ± standard deviation if they were normally dis- tributed and as the geometric mean with the 5th and 95th percentile boundaries for log10-transformed variables.
For comparisons between ethnic groups, independent T- tests were used. The proportions were determined with cross-tabs, with signiﬁcant differences indicated by Chi- square tests and presented as numbers and percentages. The correlations of the uACR with plasma and urinary
NO-related markers were tested using Pearson correlations. Standard multiple regression analyses were conducted with the uACR as a dependent variable, which was tested separately for its association with plasma and urinary NO- related markers. The covariates included in the multiple regression models included age, sex, waist-to-hip ratio, total cholesterol, eGFR, ofﬁce systolic blood pressure, MDA, smoking, glycated hemoglobin, and CK levels.

Results

The general characteristics of the study population are presented in Table 1. The black group had lower values for body composition measures, such as waist circumference, body height, and BMI (all p ≤ 0.002), than the white group, while body weight was comparable between the groups (p = 0.36). Diastolic blood pressure, central systolic blood pressure, and the MDA and CK levels were higher in the black group (all p ≤ 0.001) than in the white group. In addition, the mean uACR was similar (p = 0.34) between the groups.
When comparing NO-related markers (Table 2), the black group presented with lower urinary nitrate (by −15%) and UNOxR values (by −18%) (both p ≤ 0.001) and higher plasma (by +13%) and urinary (by +3.7%) ADMA values (both p ≤ 0.033). However, both the plasma (by −5.2%) and urinary (by −4.1%) SDMA values (both p ≤ 0.022) were lower in the black group than in the white group. No dif- ferences were observed for plasma and urinary hArg (both p ≥ 0.81). An interaction of ethnicity with the associations

Multiple regression analyses of the association of the uACR with a creatinine- corrected urinary homoarginine (hArg), b plasma hArg, and c the urinary nitrate-to-nitrite ratio in the population stratiﬁed by ethnicity the uACR with both plasma (all p ≤ 0.002) and urinary (all p ≤ 0.001) NO-related markers was observed (Supple- mentary Table 1).

We performed single regression analyses (Supplemen- tary Table 2) that showed that the uACR was associated inversely with the plasma (r = −0.176; p = 0.006)and urinary (r = −0.100; p = 0.049) hArg and UNOxR (r = −0.169; p = 0.001) values in the black group. In addition, the uACR was positively associated with urinary nitrate (r = 0.139; p = 0.008) in the white group.
In the partial regression analyses (Supplementary Table 3) (adjusted for age, sex, and BMI), we found inverse correlations of the uACR with the plasma hArg (r = −0.178; p = 0.006) and urinary hArg (r = −0.139; p = 0.007) and UNOxR (r = −0.172; p = 0.001) values in the black group only. uACR was positively associated with urinary nitrate in the white group (r = 0.124, p = 0.018). Noticeably, CK levels were inversely associated with urinary hArg (r = −0.413; p = 0.012) in the black group only. The oxi- dative stress biomarker MDA was inversely associated with the UNOxR in the black (r = −0.319; p < 0.001) and white groups (r = −0.204; p < 0.001).
In the multivariable-adjusted regression analysis (Fig. 1), we observed consistent inverse associations between the uACR and both plasma (adj. R2 = 0.066; β = −0.209; p = 0.005) and urinary (adj. R2 = 0.066; β = −0.149; p = 0.010) hArg as well as between the uACR and the UNOxR (adj. R2 = 0.060; β = −0.122; p = 0.031).

Discussion

In the present exploratory study involving a healthy cohort of both black and white patients, we compared plasma and urinary NO-related markers along with the CK levels and determined the associations with the uACR, which is an early marker of endothelial dysfunction [39]. The NO- related markers included the NOS substrates Arg and hArg [12, 17], the NOS inhibitors ADMA and SDMA [14, 40], urinary nitrate, which is a major NO metabolite reﬂecting whole-body NO synthesis [41], and urinary nitrite, which is a measure of the loss of circulating NO bioactivity [27]. We also measured urinary MDA, which is a measure of kidney- related oxidative stress [42]. We observed an overall less favorable NO proﬁle and elevated oxidative stress in the black cohort. Although the uACR did not differ between the black and white groups, the uACR was found to be inde- pendently and inversely associated with the UNOxR, plasma hArg and urinary hArg in the black cohort only. In addition, the plasma CK levels were inversely associated with urinary hArg in the black group only.
The results of our study are in line with those of a previous bi-ethnic study on black and white children, which found that black boys presented lower (by −35%) UNOxR levels than white boys [27]. The reduced urinary nitrate and UNOxR (by −18%) levels in the young black adults in the present study illustrate that whole-body NO synthesis is reduced in black individuals, with the dif- ference between black and white individuals appearing to
decrease with age. This may be attributable to genetic differences in the enzymes involved in the Arg/NO pathway, including NOS, AGAT, PRMT, and DDAH, as well as in the renal carbonic anhydrases (CA) and anion transporters involved in nitrite excretion and reabsorption from the primary urine, as previously reported [27, 43]. In addition to reduced urinary nitrate and UNOxR values, the black group also displayed increased plasma and urinary levels of Arg and ADMA, which are an endogenous NOS substrate and inhibitor, respectively. Studies have repor- ted increased Arg [20, 44] and ADMA levels [26] in black adults; however, other studies have reported ﬁndings of both increased Arg levels and increased ADMA levels in the same study population [28, 45]. The metabolic fate of Arg is complex and involves the arginase-catalyzed hydrolysis of Arg to generate ornithine, thus potentially decreasing Arg concentrations and local NO synthesis [46]. Animal studies have suggested that the expression of arginase may be elevated in hypertensive rats [47, 48]. However, the bi-ethnic differences in the Arg and orni- thine/citrulline concentrations observed in our study were relatively small, especially with respect to the differences in whole-body NO synthesis. Therefore, differences in arginase expression and activity are probably unlikely to have affected NO synthesis from Arg in our study. The ethnic disparities in regard to the NO proﬁle of our bi- ethnic cohort seem to be multifactorial and include genetic and environmental factors, which, however, are unknown. Since decreased NO synthesis is associated with hypertension [49], the decrease in NO synthesis found in the black group in this study could be responsible for the increased susceptibility of the population to the development of hypertension later in life.

In the ﬁrst instance, the uACR is an indicator of kidney function, primarily that of the glomerulus [8]. However, the uACR is also generally accepted to be an indicator of car- diovascular disease [9] and early endothelial dysfunction [39]. In our study, we found inverse associations of the uACR with the UNOxR and hArg (both in plasma and in urine) in the black group only, although both groups pre- sented with relatively similar UNOxR values. Such associa- tions have not been previously described in a black population and may suggest the considerable contribution of the kidney to these ethnic differences. Indeed, the black group presented with higher (by +10%) eGFR values compared with the white group of the cohort. The decreased UNOxR values measured in the black young adults in this study may indicate a relative increase in the loss of circu- lating nitrite, which is an important reservoir of NO bioactivity, due to attenuated CA activity in the proximal tubule of the nephron. Interestingly, the attenuation of renal CA activity is more pronounced in childhood [27] than in adulthood in black individuals.

The inverse association of plasma CK with urinary hArg (r = −0.413; p = 0.012) but not with plasma hArg in the black group only may suggest the considerable contribution of kidney function to this association. By comparing the (h) Arg level with the ADMA plasma and SDMA urine con- centrations, the urinary hArg content was found to be relatively low (Table 2). This suggests the occurrence of considerable tubular reabsorption of hArg. CK expressed in tubular cells of the kidney provides the ATP necessary for Na+/K+-ATPase activity, which drives several tubular transport mechanisms, including Na+ reabsorption in
Henle’s loop and the distal tubule [50, 51]. In a small cohort of African and European young men, plasma CK was found to be inversely associated with Na+ excretion [51].
Increased CK activity has been observed in people from the sub-Saharan African region and has been discussed as a factor contributing to hypertension in individuals of this ethnicity [23, 24].
Our large study conﬁrms the increased (by +10%) CK levels in black individuals. The increased urinary MDA excretion rate (by +19%) measured in the black group may be an indication that the kidneys of the young black adults of this study may have also been subjected to higher oxi- dative stress than the kidneys of their white counterparts. Elevated oxidative stress may in itself reduce NO bioa- vailability by increasing NO inactivation, as seen in Afri- cans [52] and Europeans [53]. Not only does an increase in oxidative stress damage the endothelium by impairing endothelium-dependent vascular relaxation (diminished NO), it also increases vascular contractile activity, which may result in hypertension [54]. These ﬁndings further reiterate that an increase in oxidative stress (MDA) [20], combined with decreased renal and systemic nitrite levels, may reduce NO bioavailability in the black population, which may contribute to structural changes in blood vessels and ultimately result in the early onset of hypertension.
The inverse association of plasma CK with urinary hArg may indicate that there is a mutual interaction between CK and AGAT in the mitochondria and cytoplasm of kidney cells. This is expected because AGAT not only produces hArg but also guanidino acetic acid (GAA). GAA is further converted to creatine, which, in turn, is the substrate of CK and an inhibitor of the AGAT-catalyzed synthesis of hArg and GAA [17, 55]. Notably, hArg has been shown to be associated with many human diseases. In particular, low circulating and excretory levels of hArg are associated with all-cause mortality [17, 56]. However, the biological activities of hArg remain almost entirely unknown. The kidney has been reported to be a major contributor to cir- culating and urinary hArg [57–59], underlining the major importance of renal AGAT activityin humans. In the pre-
sent study, we did not ﬁnd differences in the plasma and urinary hArg levels between black and white young adults.
In the healthy young adults in this study, both plasma and urinary hArg levels were closely comparable to those reported for healthy humans [56]. However, the disparate associations of urinary hArg with CK and the uACR indi- cate the occurrence of ethnic differences in these bio- markers, which presumably are related to kidney physiology.

This explorative study must be interpreted within the context of its strengths and limitations. The study was well planned and executed under strict conditions. As our population included participants from the North-West Pro- vince of South Africa, it is not representative of the popu- lation as a whole. The study is limited by its cross-sectional design; hence, we were unable to investigate the precise mechanisms and causal relationships. The participants in our study did not observe strict abstinence from a nitrate- rich diet. Dietary nitrate may have contributed to some degree to urinary nitrate but not to other biomarkers, including ADMA and hArg. Forthcoming studies on the potential involvement of Arg/NO and related pathways need to include a nitrate-poor dietary protocol.

To conclude, young black asymptomatic adults partially differ from their white counterparts with respect to various biomarkers and some of the associations between these biomarkers. Reduced urinary nitrate excretion indicates decreased whole-body NO synthesis, and a reduced UNOxR indicates renal CA-dependent loss of circulating nitrite, which is a major reservoir of NO; increased plasma ADMA levels are indicative of the elevated inhibition of NOS activity. These occurrences and the inverse associations, notably those with the uACR, which is a biomarker of early endothelial dysfunction, plasma CK, urinary MDA, which is a biomarker of kidney-associated oxidative stress, and urinary hArg, which is an endogenous substance with lar- gely unknown biological activities, were observed only in the black cohort in the study, suggesting that there were ethnicity-dependent differences in several overlapping Arg- involving pathways, i.e., pathways involving Arg/NO, AGAT, arginase, CK, and oxidative stress. Our study sug- gests that these differences originate in the cardiovascular and renal systems. Young black individuals seem to show increased susceptibility to the development of premature hypertension later in life.

Acknowledgements The authors are grateful to all individuals who participated voluntarily in the African-PREDICT study. The dedica- tion of the support and research staff as well as the students at the Hypertension Research and Training Clinic at the North-West Uni- versity (Potchefstroom campus) are also duly acknowledged.

Funding The research funded in this manuscript is part of an ongoing research project ﬁnancially supported by the South African Medical Research Council (SAMRC) with funds from the National Treasury under the Economic Competitiveness and Support Package; the South African Research Chairs Initiative (SARChI) of the Department ofScience and Technology and National Research Foundation (NRF) of South Africa (GUN 86895); SAMRC with funds received from the South African National Department of Health, GlaxoSmithKline R&D (Africa Non-Communicable Disease Open Lab grant), the UK Medical
Research Council with funds from the UK Government’s Newton Fund; and corporate social investment grants from Pﬁzer (South
Africa), Boehringer-Ingelheim (South Africa), Novartis (South Africa), the Medi Clinic Hospital (South Africa), and kind contribu- tions from the Roche Group (South Africa). Any opinions, ﬁndings, conclusions, or recommendations expressed in this material are those of the authors, and therefore, the NRF does not accept any liability in this regard.

Compliance with ethical standards

Conﬂict of interest The authors declare that they have no conﬂict of interest.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations.

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