Urinary excretion of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine is positively related to nitric oxide level in tissues of normotensive and hypertensive rats

Nitric oxide (NO) is one of the gaseous transmitters which play a very important role in the regulation of the circulatory system. Decreased NO availability is associated with hypertension, cardiovascular and kidney diseases. Endogenous NO is generated enzymatically by nitric oxide synthase (NOS) depending on the availability of the substrate, cofactors, or presence/absence of inhibitors, such as asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). The objective of this study was to evaluate the potential relationship between NO level in rat tissues (heart and kidneys) and the concentrations of endogenous metabolites related to NO in plasma and urine. The experiment was carried out with 16- and 60-week-old male Wistar Kyoto (WKY) and age-matched male Spontaneously Hypertensive Rats (SHR). NO level in tissue homogenates was determined by the colorimetric method. RT-qPCR was used to verify the expression of the eNOS (endothelial NOS) gene. Plasma and urine concentrations of arginine, ornithine, citrulline, and dimethylarginines were examined by the UPLC-MS/MS method. 16-week-old WKY rats had the highest tissue NO and plasma citrulline levels. Furthermore, 16-week-old WKY rats showed higher urinary excretion of ADMA/SDMA compared to other experimental groups, however, plasma concentrations of arginine, ADMA, and SDMA were comparable between the groups. In conclusion, our research shows that hypertension and aging decrease tissue NO levels and are associated with reduced urinary excretion of NOS inhibitors, i.e., ADMA and SDMA.


Introduction
In the past, nitric oxide (NO) was considered a toxic gas associated with environmental pollution . However, it is now known that NO is one of the most potent physiological vasodilators that plays a very important role in the regulation of circulatory system (Li et al. 2009;Farah et al. 2018;Gantner et al. 2020). Endogenous NO is generated enzymatically by oxidation of l-arginine to l-citrulline ( Fig. 1) in the presence of numerous cofactors. This reaction can be catalyzed by one of the three well-recognized isoforms of nitric oxide synthase (NOS): neuronal (nNOS), inducible (iNOS) or endothelial (eNOS) (Farah et al. 2018;Wu et al. 2018;Gheibi et al. 2020). eNOS occurring in vascular endothelial cells is the main source of nitric oxide under physiological conditions (Gkaliagkousi et al. 2009) and is characterized by constitutive activity (Moroz and Kohn 2011;Liu et al. 2018). Moreover, NO can be formed Handling editor: D. Tsikas. by non-enzymatic nitrate/nitrite reduction (Gheibi et al. 2020).
The bioavailability of l-arginine, as well as its metabolites, plays an essential role in the regulation of NO production. Methylation of arginine residues in proteins by protein arginine N-methyltransferases leads to the formation of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) (Böger 2003;Lin et al. 2016;Hannemann et al. 2021) (Fig. 1). Both ADMA and SDMA are inhibitors of NOS activity but have different inhibitory potential (Tsikas et al. 2018). SDMA is eliminated from the body by urinary excretion, while ADMA is removed mainly enzymatically and renal excretion is less important (Oliva-Damaso et al. 2019). As a result of enzymatic degradation of ADMA by dimethylarginine dimethylaminohydrolase (DDAH), citrulline and dimethylamine are formed (Böger 2003;Lin et al. 2016;Hannemann et al. 2021). In turn, l-arginine hydrolysis by the enzyme arginase leads to the formation of ornithine (Cederbaum et al. 2004), which can then be enzymatically converted into citrulline. Subsequently, citrulline can be reused to produce arginine via the argininosuccinate pathway (Lin et al. 2016) (Fig. 1).
Nitric oxide has been shown to play a cardio-protective role by regulating endothelium-dependent vasorelaxation via the soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway (Ahmad et al. 2018;Ataei Ataabadi et al. 2020). Moreover, nitric oxide mediates angiogenesis, inhibits platelet aggregation and vascular smooth muscle cells proliferation (Gkaliagkousi et al. 2009;Farah et al. 2018). Furthermore, it has anti-adhesive properties-inhibits migration and adhesion of leukocytes and monocytes to the endothelium, and antioxidant properties-reduces production of superoxide radicals and oxidation of LDL cholesterol. Therefore, any reduction in physiological NO levels can lead to hypertension and/or atherosclerosis (Böger 2003;Gkaliagkousi et al. 2009). Additionally, nitric oxide controls renal hemodynamics and promotes diuresis and urinary excretion of sodium, as well as adaptation of kidneys to dietary salt intake (Gheibi et al. 2020). Therefore, it is postulated that renal NO production deficiency is the main mechanism for the development of systemic hypertension (Ahmad et al. 2018).
Hypertension and its complications are the leading causes of death and disability in aging societies. Spontaneously Hypertensive Rats (SHRs) are the most commonly used animal model to study hypertension. SHRs start to develop hypertension between the 4-6th week of age, and by 10 weeks their arterial blood pressure is 30% higher than in Wistar Kyoto (WKY) rats. 12-month-old SHRs showed characteristics of heart failure with preserved ejection fraction and compromised diastolic function (Huc et al. 2018). The SHR strain is derived from normotensive WKY rats (Okamoto and Aoki 1963).
The aim of this study was to evaluate the potential relationship between NO level in tissues collected from normotensive or hypertensive rats of different ages and the concentrations of NO-related endogenous metabolites in plasma and urine, which can provide a potential therapeutic approach to prevent the progression of hypertension via the NO pathway.

Blood and urine samples collection, tissue collection
The rats were housed for 2 days in metabolism cages to evaluate 24-h water balance and to collect urine for biochemical analysis. The following day, animals were anesthetized with an intraperitoneal injection of urethane (1.5 g/kg bw). After collecting blood from the right ventricle of the heart, the rats were killed by decapitation. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes, and centrifuged for 15 min at 2500×g and 4 °C. During dissection, the hearts and kidneys were taken. Tissues, centrifuged plasma, and urine were immediately frozen in liquid nitrogen and kept at − 80 °C for further procedures.

Nitric oxide level in tissue homogenates
To determine the total nitrate/nitrite concentration (a marker of nitric oxide level), rat hearts and kidneys were homogenized in ice-cold phosphate buffered saline pH 7.4 (1:5, w/v) for 1 min at 8000-9500 rpm using a blender homogenizer. The homogenates were centrifuged at 10,000×g for 20 min, and then the supernatants were ultra-filtered using 30 kDa molecular weight cut-off filters for 20 min. Total nitrate/nitrite concentration was determined using a colorimetric method according to the manufacturer's instructions (780001, Cayman Chemicals, Ann Arbor, MI, United States). The nitric oxide level was expressed as µM of total NO 3 − /NO 2 − concentration per 1 mg of protein.

Protein level in tissue homogenates
To evaluate protein content, tissue samples were weighed and homogenized for 1 min at 8000-9500 rpm in ice-cold 0.1 M phosphate buffer pH 7.5 (1:4, w/v), then centrifuged for 10 min at 1600×g. Protein concentration in supernatants was determined by the method of Lowry et al. (1951) using crystalline BSA as a standard.

Expression of eNOS gene
Total RNA was extracted from tissues using the Tri Reagent, according to the manufacturer's protocol. Extracted RNA was suspended in nuclease-free water. Agarose gel electrophoresis and spectrophotometric measurement with NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) were performed to assess the integrity and purity of the RNA samples. The RNA solutions were kept at − 80 °C until further procedures. The total RNA from tissue samples was reverse-transcribed using the GoScript™ Reverse Transcriptase Kit according to the manufacturer's instructions (Promega, Madison, WI, USA). The detailed mix composition and reaction conditions were described in the previous study (Szlęzak et al. 2021). The cDNA solutions were kept at − 20 °C. eNOS gene expression was determined by quantitative real-time PCR (qPCR) analysis using the MiniOpticon™ System (Bio-Rad, Hercules, CA, USA), Fast SYBR Green qPCR Master Mix (EURx, Gdańsk, Poland), and specific primers (Sigma-Aldrich, Darmstadt, Germany). qPCR amplifications consisting of 40 cycles of denaturation and annealing/extension were performed according to the protocol provided by the manufacturer. EEF1A1 (eukaryotic translation elongation factor 1 alpha 1) gene was used as a reference gene. The primer sequences were as follows: eNOS forward 5′-TAA CTA GAC TGG GAG GGA GTCA-3′ and eNOS reverse 5′-AAA GCA TAC GAA GAG GGC AG-3′ (product size 88 bp), EEF1A1 forward 5′-TTT CGC AAC GGG TTT GCC -3′ and EEF1A1 reverse 5′-GCC GGA ATC TAC GTG TCC AA-3′ (product size 121 bp) (Szlęzak et al. 2022). eNOS primers were designed using the NCBI OMIM database and NCBI Primer-BLAST tool. The mRNA expression levels of target gene were calculated by the 2 −ΔCq method.

Level of NO-related pathway endogenous metabolites and dimethylarginines
The UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry) method was used to determine plasma and urine concentrations of arginine, ornithine, citrulline, and asymmetric and symmetric dimethylarginine. The analysis was performed by a Waters Acquity ultra-performance liquid chromatograph coupled with a Waters TQ-S triple-quadrupole mass spectrometer. Separations were carried out with a Waters HILIC column (1.7 μm, 2.1 mm × 50 mm). A mobile phase consisting of solvent A-water containing 0.1% ammonium hydroxide and solvent B-acetonitrile containing 0.1% formic acid was used to elute the samples. Multiple reaction monitoring (MRM) via positive electrospray ionization (ESI +) mode on a mass spectrometer was used. Samples were prepared using acetone-10 μl of the sample was mixed with 100 μl of acetone containing internal standards (isotope-labeled ADMA-D 6 , arginine-D 4 and ornithine-13 C 5 ), the injection volume was 7 μl (Gąsecka et al. 2021). The internal standard at known concentration was added to each sample (calibrators, plasma, and urine) to determine the response which is used to generate a calibration curve and quantify the concentration of the analytes. The calibration curve was generated by comparing a ratio of the analyte's peak area to the peak of the internal standard against known analyte concentration. Biological samples were compared with an obtained calibration curve and concentrations were calculated. The analyses were carried out at the Mass Spectrometry Laboratory of the Institute of Biochemistry and Biophysics Polish Academy of Sciences (Warsaw, Poland).

Statistical analysis
All results were represented as arithmetic means with standard deviations (SD). The lack of normal distribution of the data and the lack of homogeneity of variance were confirmed by the Shapiro-Wilk test and the Levene test, respectively. Statistical analysis was performed via the Mann-Whitney U test. Differences with a p value < 0.05 were considered statistically significant. Analyses were conducted using Statsoft Statistica 13 software (Tibco, Palo Alto, CA, USA).

Animal parameters
Detailed metabolic and hemodynamic rat parameters were previously described in the study by Huc et al. (2018). The following mean arterial blood pressure values ± SE were observed in anesthetized WKY rats: 83.73 ± 4.46 mmHg in 16-week-old and 81.79 ± 2.19 mmHg in 60-weekold WKY. Significantly higher values (p < 0.05 vs. age-matched WKY groups) were observed in anesthetized SHR rats: 101.6 ± 4.34 mmHg in 16-week-old and 120.98 ± 5.03 mmHg in 60-week-old SHRs. In the 16-weekold animal groups, higher food intake and stool output, and lower water intake were observed in WKY rats compared to SHRs. The body mass of these rats was similar. There were no differences in food and water intake, stool output, and body mass in the groups of 60-week-old rats. The mean 24-h urine output values ± SE were as follows: 10.14 ± 0.57 ml in 16-week-old WKY, 15.87 ± 2.33 ml in 60-week-old WKY, 15.14 ± 1.36 ml in 16-week-old SHR and 16.16 ± 1.66 ml in 60-week-old SHR (Huc et al. 2018).

NO level in tissues
Total nitrate/nitrite concentrations in tissue homogenates were used to determine nitric oxide levels in the hearts and kidneys of rats (Fig. 2). The highest levels of nitrate/nitrite were observed in the hearts ( Fig. 2A) and kidneys (Fig. 2B) of 16-week-old WKY rats, and these values were more than three times higher compared to tissues of age-matched SHRs and more than two times higher compared to tissues of 60-week-old WKY rats. In hypertensive rats, NO level in the hearts was the same in both age groups, while in the kidneys it was statistically significantly higher in 60-week-old SHRs compared to 16-week-old SHRs.

eNOS gene expression at the mRNA level
The highest level of eNOS gene expression was found in the hearts of 16-week-old hypertensive rats and this value was significantly higher than in the 60-week-old SHRs and 16-week-old WKY rats (Fig. 3A). No statistically significant changes were observed in the expression of the endothelial NOS gene in groups of normotensive rats of different age, but there was a tendency to decrease this expression in the hearts of 60-week-old animals. The expression of the eNOS gene in the kidneys of WKY rats in both age groups was low (Fig. 3B), a significantly higher expression level was found in the groups of rats with hypertension.

Plasma concentrations and urinary excretion of NO-related pathway endogenous metabolites
There were no statistically significant differences in the plasma arginine concentration of the examined groups of rats (Fig. 4A). Plasma ornithine level was significantly higher in older hypertensive rats than in age-matched WKY rats and 16-week-old SHRs. In turn, concentration of citrulline was significantly higher in the plasma of 16-week-old WKY rats compared to 60-week-old WKY and 16-week-old SHRs. Fig. 2 Total nitrate/nitrite concentrations in the hearts (A) and kidneys (B) of rats. Values represent an arithmetic mean ± SD of 6-12 repetitions for 2-4 animals per group. 16-WKY 16-week-old WKY rats, 60-WKY 60-week-old WKY rats, 16-SHR 16-week-old SHRs, 60-SHR 60-week-old SHRs. *p < 0.05 SHR vs. WKY of the same age, # p < 0.05 60-week-old vs. 16-week-old Fig. 3 Expression of the eNOS gene in the hearts (A) and kidneys (B) of rats. Values represent an arithmetic mean ± SD (n = 3 animals per group). Each value is the mean of at least two independent experiments. 16-WKY 16-week-old WKY rats, 60-WKY 60-week-old WKY rats, 16-SHR 16-weekold SHRs, 60-SHR 60-weekold SHRs. *p < 0.05 SHR vs. WKY of the same age, # p < 0.05 60-week-old vs. 16-week-old Fig. 4 Plasma concentrations (A) and urinary excretion (B) of NOrelated pathway endogenous metabolites in rats. The concentrations in the urine samples were normalized to 24-h urinary excretion. Values represent an arithmetic mean ± SD (n = 6-9 animals per group). The colored circles show the individual data values. 16-WKY 16-week-old WKY rats, 60-WKY 60-week-old WKY rats, 16-SHR 16-week-old SHRs, 60-SHR 60-week-old SHRs, ARG arginine, CIT citrulline, ORN ornithine, < LOQ below the limit of quantification. The quantification limits were 4.03 µM for arginine, 5.04 µM for ornithine, and 5.85 µM for citrulline. *p < 0.05 SHR vs. WKY of the same age, # p < 0.05 60-week-old vs. 16-week-old The amount of arginine excreted in urine during 24 h was lowest in the group of 60-week-old hypertensive rats (Fig. 4B). 16-week-old WKY rats excreted the most ornithine and this value was significantly higher compared to older WKY rats. No citrulline was detected in rats' urine.
In the studied groups, no statistically significant correlation was found between plasma concentrations and urinary excretion of arginine and ornithine (data not shown).

Plasma concentrations and urinary excretion of asymmetric and symmetric dimethylarginine
The concentrations of ADMA in plasma were similar in all groups of rats (Fig. 5A), except for the 60-week-old SHRs, which showed statistically lower levels of ADMA than age-matched WKY rats and 16-week-old SHRs. Mean plasma SDMA concentrations in normotensive and 16-week-old hypertensive rats ranged from 0.49 to 0.54 µM (Fig. 5A). In the plasma of 60-week-old hypertensive rats, a statistically significant decrease in SDMA level was observed compared to 16-week-old SHRs.
The mean 24-h urinary excretion of ADMA and SDMA was the highest in the group of 16-week-old WKY rats (Fig. 5B). However, statistically significant differences were observed only in the amount of ADMA in the urine of 16-week-old SHR compared to age-matched WKY rats. The colored circles show the individual data values. 16-WKY 16-week-old WKY rats, 60-WKY 60-week-old WKY rats, 16-SHR 16-week-old SHRs, 60-SHR 60-week-old SHRs, ADMA asymmetric dimethylarginine, SDMA symmetric dimethylarginine. The quantification limits were 0.03 µM for ADMA and 0.02 µM for SDMA. *p < 0.05 SHR vs. WKY of the same age, # p < 0.05 60-week-old vs.

Discussion
Our research shows that hypertension and aging decrease tissue NO levels and reduce urinary excretion of NOS inhibitors, i.e., ADMA and SDMA, in rats. Furthermore, it provides original data on changes in the expression of the eNOS gene at the mRNA level in rat tissues depending on age and absence/presence of hypertension. The experiments confirmed that NO level undergoes a reduction in heart and kidney homogenates from hypertensive and 60-week-old normotensive rats. These changes occurred independently of changes in eNOS gene expression. In the cardiovascular system, all three NOS isoforms have regulatory functions. nNOS is responsible for central regulation of blood pressure, iNOS plays a protective role in septic shock, and eNOS determines vasodilation, vasoprotection, and prevention of atherosclerosis (Förstermann and Sessa 2012). The crucial role of eNOS in the pathogenesis of hypertension has been confirmed by the observation that eNOS-knockout mice develop hypertension (Huang et al. 1995;Shesely et al. 1996). Nevertheless, there is a lack of research on the level of eNOS gene expression in tissues of normotensive and hypertensive rats, and reports on the effect of age on this expression in rats' vessels are contradictory (El Assar et al. 2013). In our study, the expression of eNOS in SHRs tissues was (depending on the type of tissue and the age of the animals) higher or similar to that in WKY rats. Previous studies using human and bovine aortic endothelial cells showed that eNOS expression increases at the mRNA level under the influence of reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) (Drummond et al. 2000). ROS are important modulators in cardiovascular diseases and hypertension (Davalli et al. 2016)-their increased levels in the myocardium in SHRs compared to WKY rats have been experimentally demonstrated (Shi et al. 2007). However, the level of eNOS gene expression does not necessarily translate into the amount of nitric oxide formed, since the activity of NOS is regulated by many cofactors. The shortage of cofactors leads to eNOS uncoupling and the formation of superoxide anions (Münzel et al. 2005), which in turn react with NO and generate highly reactive peroxynitrite (ONOO -) associated with age-related vascular dysfunction (El Assar et al. 2013). Nitric oxide deficiency is associated with increased blood pressure, as well as with cardiovascular and kidney diseases (Lin et al. 2016). Decreased endogenous NO concentrations have been reported in the plasma of patients with essential hypertension (Node et al. 1997) and in the urine of patients with chronic kidney disease (Schmidt and Baylis 2000).
The levels of metabolites, such as arginine, citrulline, and ornithine, are closely related to the maintenance of NO homeostasis (Fig. 1). The important source of arginine for nitric oxide synthesis in endothelial cells of blood vessels is extracellular arginine, present in plasma (Wu and Meininger 2002). In our experiment, no statistically significant changes were observed in mean plasma arginine concentrations of hypertensive rats compared to normotensive rats, although there was a trend toward a decrease in mean arginine concentrations in SHRs. The high level of citrulline observed in the plasma of 16-week-old normotensive rats was correlated with the high level of nitric oxide in the tissues of these rats. In turn, different plasma concentrations of ornithine could have been caused by the differential activity of arginase, considered to be the key regulator of NO availability, which influences NOS activity using their common substrate (Rabelo et al. 2015). White et al. (2006) showed that the aortic NO production of 3-monthold WKY rats is higher compared to 23-month-old rats with simultaneous higher NOS activity and decreased arginase activity. In turn, skeletal muscle arterioles isolated from salt-loaded hypertensive Dahl rats showed higher levels of arginase compared to low-salt normotensive controls (Johnson et al. 2005). Similarly, it was found that in diabetic hypertensive rats the activity of serum arginase was increased, and inhibition of this enzyme was associated with a reduction in diastolic blood pressure and an increased aortic NO production (El-Bassossy et al. 2012). Kondziella et al. (2008) showed that plasma levels of ornithine were higher in SHRs compared to WKY control rats. In our study, we found a significantly higher ornithine concentration in the plasma of 60-week-old hypertensive rats, which may be a determinant of arginase activity.
Previous papers have shown that increased plasma ADMA levels are associated with diseases related to endothelial dysfunction, such as cardiovascular diseases, diabetes (Tain and Hsu 2017), as well as with chronic kidney disease (Baylis et al. 2012). It is known that ADMA contributes to the development of hypertension in two ways: by inhibiting the activity of eNOS, which causes vasoconstriction, and by reducing renal NO synthesis, which may lead to suppression of sodium excretion by the kidneys (Oliva-Damaso et al. 2019). Achan et al. (2003) showed that intravenous administration of low doses of ADMA leads to the development of hypertension and cardiac dysfunction in humans. Increased plasma ADMA concentrations are considered a risk factor for premature mortality in renal transplant recipients (Frenay et al. 2015). On the other hand, high plasma SDMA levels have been associated with hypertension and hyperlipidemia in patients with rheumatoid arthritis (Chandrasekharan et al. 2018). Also, the level of urinary ADMA and/or SDMA seems to be an important parameter in the assessment of the patient's condition. The study by Said et al. (2019) showed that high urinary excretion of ADMA and SDMA in renal transplant 1 3 recipients was associated with a lower risk of cardiovascular diseases mortality. Similarly, in patients with coronary artery disease, low urinary ADMA concentrations have been associated with impaired heart function and increased mortality (Wolf et al. 2012). In our experiment, 16-week-old normotensive rats excreted the most ADMA and SDMA. However, in all experimental groups, the average amount of symmetric dimethylarginine excreted in the urine was significantly higher (29-49 times on average) than the average amount of asymmetric dimethylarginine. Previously, a comparable effect was observed in male Sprague Dawley rats, where the amount of excreted SDMA was on average 22-25 times higher than ADMA (Al Banchaabouchi et al. 2000;Matsuguma et al. 2006).
This study has shown that 16-week-old WKY rats were characterized by the highest total concentration of nitrate/ nitrite in tissue homogenates. At the same time, there was the highest 24-h urinary excretion of ADMA and SDMA in the group of these rats. On this basis, we suggest that urinary elimination of these dimethylarginines is positively related to NO level in rat tissues (Fig. 6). However, plasma concentrations of arginine, ADMA, and SDMA were comparable in all study groups. Therefore, we hypothesize that tissue protein turnover plays a key role in NO formation and ADMA/SDMA homeostasis.
In addition to the above, there is some evidence of a crosstalk between nitric oxide and hydrogen sulfide (H 2 S). Hosoki et al. (1997) showed that the simultaneous use of H 2 S and NO donors induced greater relaxation of vascular smooth muscle than one or the other molecule alone. On the other hand, hydrogen sulfide donors protected endothelial NO production under oxidative stress conditions (Al-Magableh et al. 2014). Studies in mice have shown that hydrogen sulfide therapy protects against heart failure (Kondo et al. 2013) and attenuates I/R injury (King et al. 2014) by increasing eNOS activity. In turn, Zhao et al. (2001) showed that incubation of homogenized rat vascular tissues with different doses of NO donor enhances the production of H 2 S in a concentration-dependent manner. Our current and previous examination (Szlęzak et al. 2022) indicates a correlation between the formation of nitric oxide and hydrogen sulfide in the examined tissues-their levels were reduced in the hearts and kidneys of 60-week-old normotensive rats and hypertensive rats in both age groups. The obtained results confirm the significant relationship between these two molecules under physiological conditions and provide the basis for further, more advanced research.
The limitation of the presented studies is that the individual methods used (total nitrate/nitrite determination, protein content determination) require separate material, a different tissue fragment, and different homogenate preparation procedures, which, however, should not significantly affect the final results.

Conclusions
In summary, our study shows that NO level is reduced in the hearts and kidneys of hypertensive and older normotensive rats, however, this is not dependent on eNOS gene expression. Increased urinary excretion of ADMA and SDMA in younger normotensive rats may suggest that higher elimination of ADMA and SDMA from the body increases the level of nitric oxide in rat tissues (Fig. 6). Further studies evaluating the effects of ADMA and SDMA are needed to understand the role of the methylamines in hypertension and aging-related processes.
Author contributions DS, MU, AD and ES collected the data. DS and MU analyzed the data. DS wrote the manuscript. MU and MW supervised the study and revised the paper. All authors have read and agreed to the published version of the manuscript. Fig. 6 Proposed dependence between urinary excretion of dimethylarginines and NO level in tissues of normotensive/ hypertensive rats of different ages. ADMA asymmetric dimethylarginine, NO nitric oxide, SDMA symmetric dimethylarginine Funding This work was supported by the Polish Ministry of Science and Higher Education (Grant no. N41/DBS/000435) and the Polish National Science Centre (Grant no. 2020/37/B/NZ5/00366).

Data availability
The authors confirm that the data supporting the findings of this study are included in the article.

Declarations
Competing interests The authors declare no competing interests.

Conflict of interest
The authors declare no conflict of interest.
Ethical approval Biological material was collected from control rats in the study by Huc et al. (2018) approved by the I Local Ethical Committee in Warsaw (permission: 100/2016). All animal procedures were in accordance with the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The conducted experiments are not a clinical trial.
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