Abstract
High salt intake is a risk factor for hypertension, which can potentially lead to erectile dysfunction (ED); however, the underlying pathological mechanisms remain unclear.
To investigate whether erectile function is directly impaired by high salt intake and whether selective inhibition of mineralocorticoid receptor (MR) could provide protection from ED.
6-week-old male Dahl salt-sensitive rats were randomly divided into 3 groups: normal diet (0.3% NaCl; control, n = 8), high-salt diet (8% NaCl; HS, n = 8), and high-salt diet plus eplerenone (HS + EPL, n = 11). HS + EPL rats were orally administered daily doses of EPL (75 mg/kg) for 6 weeks; control and HS rats received purified water on the same schedule.
At the end of the study period, erectile function was evaluated by measuring intracavernosal pressure and mean arterial pressure after cavernous nerve stimulation. Serum levels of asymmetric dimethylarginine and L-arginine were determined using ultraperformance liquid chromatography–tandem mass spectrometry. Quantitative PCR was used to assess the expression of MR, inflammation, and oxidative stress markers (nicotinamide adenine dinucleotide phosphate oxidase-1/4, p22phox, interleukin-6, and superoxide dismutase-1), and protein arginine N-methyltransferase-1.
The intracavernosal pressure/mean arterial pressure ratio was significantly lower, whereas systolic blood pressure, MR expression, serum asymmetric dimethylarginine levels, oxidative stress, and levels of inflammatory biomarkers were significantly higher in HS rats than in control rats (P < .05). EPL administration significantly improved each of these parameters except systolic blood pressure and MR expression. No significant intergroup differences were observed for L-arginine and superoxide dismutase-1 levels.
Our results provide a rationale for the need of salt restriction and the use of selective MR inhibitors in prophylaxis or treatment of ED in men consuming a high-salt diet.
We are the first to report that the adverse impact of high salt intake on erectile function is mediated via MR activation, independent of its effect on blood pressure. A major limitation of this study is that responses of salt-resistant rats were not studied.
High salt intake directly impaired erectile function in Dahl salt-sensitive rats, whereas selective MR inhibition ameliorated this effect.
Introduction
Blood pressure is regulated through the renin-angiotensin-aldosterone-system in the kidney and involves activation of the mineralocorticoid receptor (MR) by aldosterone. The amount of serum aldosterone is adjusted according to the amount of sodium chloride derived from the diet.1–4 In contrast, high salt intake leads to aberrant MR activation. A high-salt diet is a risk factor for hypertension,5 which itself is a major risk factor for cardiovascular disease, kidney disease, and stroke. Various mechanisms for salt-induced hypertension have been proposed, including volume expansion, modified renal function, sodium imbalance, impairment of the renin-angiotensin-aldosterone-system and the associated receptors, central stimulation of the sympathetic nervous system, and inflammation.1,6 Several epidemiologic studies and meta-analyses reported that a high-salt diet causes cardiovascular disease7,8 through abnormal activation of the MR,9 independently of its effect on blood pressure.1 MR blockade improved the prognosis after heart failure in Randomized Aldactone Evaluation Study (RALES)10 and Eplerenone Post–Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS).11 The potential applications of selective MR antagonism in the treatment of erectile dysfunction have also been explored.12,13
In this study, we investigated whether high salt intake directly impaired erectile function and whether MR inhibition could ameliorate this effect.
Materials and methods
Animals
6-week-old male Dahl salt-sensitive (Dahl-S) rats were purchased from Japan SLC, Inc (Hamamatsu, Japan).14 All experimental protocols were approved by the appropriate ethics review board (H25-P-09, Tokudōbutsu11085) and conducted in accordance with institutional standards for the care and use of animals.
Treatment Protocols
The rats were randomly divided into 3 groups: normal diet (0.3% NaCl; control, n = 8), high salt diet (8% NaCl; HS, n = 8), and high salt diet plus eplerenone (HS + EPL, n = 11). HS + EPL rats were administered EPL (Pfizer Inc, NY) dissolved in purified water every day for 6 weeks (75 mg/kg/day, po); control and HS rats received purified water on the same schedule. Systemic arterial blood pressure of the rats was measured before and after treatment, using a noninvasive automatic device (BP-98A-L, Softron, Tokyo, Japan).15 The rats were maintained at 37°C during the measurements by means of a warmer (THC-31, Softron). Each measurement was performed 3 times per rat (while awake), and the mean value was used for analysis.
Blood Sample Collection
At the end of the treatment period (rat age = 12 weeks), blood samples were obtained from the rats via the vena cava. The blood was allowed to clot before the serum was separated by centrifugation (800 × g for 20 min at 4°C) and stored at −80°C until analysis.
Quantification of Serum Asymmetric Dimethylarginine and L-Arginine
Asymmetric dimethylarginine (ADMA) and L-arginine (L-Arg) levels were measured as previously reported.16–19 Briefly, aliquots of standards or serum were transferred into microcentrifuge tubes, and internal standards (D6-ADMA and D6-L-Arg) were added to each tube. Deproteinized samples were separated via ultraperformance liquid chromatography–tandem mass spectrometry (Acquity UPLC-MS/MS; Waters, Milford, MA), using an Acquity UPLC BEH Amide 1.7 μm column (2.1 × 100 mm; Waters) for separation in water containing 0.08% ion-pair reagent (IPCC-MS; GL Science) and acetonitrile containing 1% formic acid as the mobile phase. Serum sodium levels were measured by Fukuyama Rinsho Co, Ltd (Fukuyama, Japan).
Assessment of Erectile Function
The cavernous nerve was electrically stimulated, and intracavernosal pressure (ICP) was measured according to our previously described method.17–21 In brief, rats from each group were anaesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg); anesthesia was maintained using isoflurane (Mylan, Canonsburg, PA). The carotid artery was cannulated to allow continuous monitoring of the mean arterial pressure (MAP). The left crus of the corpus cavernosum (CC) was cannulated with a 23-G needle to allow continuous ICP monitoring after being connected to a pressure transducer with polyethylene-50 tubing coated with 50 U/L of heparin. The pressure transducer was connected through a transducer amplifier to a data acquisition board (PowerLab 2/26, AD Instruments Pty., New South Wales, Australia). Stainless steel bipolar wire electrodes (Unique Medical, Osaka, Japan) and a pulse generator (Nihon Kohden, Tokyo, Japan) were used to electrically stimulate penile erections. The following stimulation parameters were judged to cause a maximal reaction: 10 V, 20 Hz, a square wave duration of 5 ms, for 1-minute duration.17–21 Response parameters were calculated using Chart & Scope software (ADInstruments). Erectile function was evaluated on the basis of the maximum ICP/MAP ratio, as ICP is influenced by systemic arterial pressure.
Tissue Sample Collection
After erectile response measurements, samples of rat penile shafts were harvested for quantitative PCR (qPCR) analysis. The urethra and blood vessels were removed from the penis, and the CC was collected. All samples were stored at −80°C until analysis.
Quantitative PCR
qPCR analysis was performed as previously reported.17,20,21 Total RNA was extracted from CC samples using the Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions, before 1 μg of extracted RNA was reverse-transcribed into cDNA using the ReverTra Ace-α kit (Toyobo, Osaka, Japan). The cDNA served as the template for qPCR using the Power SYBR PCR Master Mix (Applied Biosystems, Tokyo, Japan). The primer sequences are shown in Table 1 . Amplification and detection were performed on the ABI 7300 system (Applied Biosystems). Gene expression levels of MR, p22phox, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1, NADPH oxidase-4, interleukin (IL)-6, superoxide dismutase-1 (SOD-1), and protein arginine N-methyltransferase-1 (PRMT-1) were quantified relative to β-actin expression using the comparative CT method. All measurements were performed in triplicate.
Quantitative PCR primer sequences
| mRNA | Sequence | |
|---|---|---|
| MR | Forward | 5′-TTGAGAATGTCGCGTCCCTC-3′ |
| Reverse | 5′-GCAGTGGGATCGTCACTGG-3′ | |
| p22phox | Forward | 5′-TACCTGACCGCTGTGGTGAAG-3′ |
| Reverse | 5′-GCAGTAAGTGGAGGACAGCCC-3′ | |
| NADPH oxidase-1 | Forward | 5′-GCTGGCCTTACTGGAGTGGTC-3′ |
| Reverse | 5′-TCCTGCGGATAAACTCCATAGC-3′ | |
| NADPH oxidase-4 | Forward | 5′-CTGTCCTGAACCTCAACTGCAG-3′ |
| Reverse | 5′-TGTGATCCGCGAAGGTAAGC-3′ | |
| IL-6 | Forward | 5′-CAAGAGACTTCCAGCCAGTTGC-3′ |
| Reverse | 5′-TGTTGTGGGTGGTATCCTCTGTG-3′ | |
| SOD-1 | Forward | 5′-GGGGACAATACACAAGGCTGT-3′ |
| Reverse | 5′-CCTTTCCAGCAGCCACATTG-3′ | |
| PRMT-1 | Forward | 5′-TTGACTCCTATGCCCACT -3′ |
| Reverse | 5′-CCACATCCAGCACCACC -3′ | |
| β-actin | Forward | 5′-TGTGTGGATTGGTGGCTCTATC-3′ |
| Reverse | 5′-CATCGTACTCCTGCTTGCTGATC-3′ | |
| mRNA | Sequence | |
|---|---|---|
| MR | Forward | 5′-TTGAGAATGTCGCGTCCCTC-3′ |
| Reverse | 5′-GCAGTGGGATCGTCACTGG-3′ | |
| p22phox | Forward | 5′-TACCTGACCGCTGTGGTGAAG-3′ |
| Reverse | 5′-GCAGTAAGTGGAGGACAGCCC-3′ | |
| NADPH oxidase-1 | Forward | 5′-GCTGGCCTTACTGGAGTGGTC-3′ |
| Reverse | 5′-TCCTGCGGATAAACTCCATAGC-3′ | |
| NADPH oxidase-4 | Forward | 5′-CTGTCCTGAACCTCAACTGCAG-3′ |
| Reverse | 5′-TGTGATCCGCGAAGGTAAGC-3′ | |
| IL-6 | Forward | 5′-CAAGAGACTTCCAGCCAGTTGC-3′ |
| Reverse | 5′-TGTTGTGGGTGGTATCCTCTGTG-3′ | |
| SOD-1 | Forward | 5′-GGGGACAATACACAAGGCTGT-3′ |
| Reverse | 5′-CCTTTCCAGCAGCCACATTG-3′ | |
| PRMT-1 | Forward | 5′-TTGACTCCTATGCCCACT -3′ |
| Reverse | 5′-CCACATCCAGCACCACC -3′ | |
| β-actin | Forward | 5′-TGTGTGGATTGGTGGCTCTATC-3′ |
| Reverse | 5′-CATCGTACTCCTGCTTGCTGATC-3′ | |
IL-6 = interleukin 6; MR = mineralocorticoid receptor; NADPH = nicotinamide adenine dinucleotide phosphate; PRMT = protein arginine N-methyltransferase; SOD = superoxide dismutase.
The following thermal cycler conditions were used: 2 min at 50°C; 10 min at 95°C; 40 cycles of 95°C for 15 s and 60°C for 1 min; 15 s at 95°C, 15 s at 60°C, and 15 s at 95°C. Primer specificity was confirmed via dissociation curve analysis after qPCR amplification.
Quantitative PCR primer sequences
| mRNA | Sequence | |
|---|---|---|
| MR | Forward | 5′-TTGAGAATGTCGCGTCCCTC-3′ |
| Reverse | 5′-GCAGTGGGATCGTCACTGG-3′ | |
| p22phox | Forward | 5′-TACCTGACCGCTGTGGTGAAG-3′ |
| Reverse | 5′-GCAGTAAGTGGAGGACAGCCC-3′ | |
| NADPH oxidase-1 | Forward | 5′-GCTGGCCTTACTGGAGTGGTC-3′ |
| Reverse | 5′-TCCTGCGGATAAACTCCATAGC-3′ | |
| NADPH oxidase-4 | Forward | 5′-CTGTCCTGAACCTCAACTGCAG-3′ |
| Reverse | 5′-TGTGATCCGCGAAGGTAAGC-3′ | |
| IL-6 | Forward | 5′-CAAGAGACTTCCAGCCAGTTGC-3′ |
| Reverse | 5′-TGTTGTGGGTGGTATCCTCTGTG-3′ | |
| SOD-1 | Forward | 5′-GGGGACAATACACAAGGCTGT-3′ |
| Reverse | 5′-CCTTTCCAGCAGCCACATTG-3′ | |
| PRMT-1 | Forward | 5′-TTGACTCCTATGCCCACT -3′ |
| Reverse | 5′-CCACATCCAGCACCACC -3′ | |
| β-actin | Forward | 5′-TGTGTGGATTGGTGGCTCTATC-3′ |
| Reverse | 5′-CATCGTACTCCTGCTTGCTGATC-3′ | |
| mRNA | Sequence | |
|---|---|---|
| MR | Forward | 5′-TTGAGAATGTCGCGTCCCTC-3′ |
| Reverse | 5′-GCAGTGGGATCGTCACTGG-3′ | |
| p22phox | Forward | 5′-TACCTGACCGCTGTGGTGAAG-3′ |
| Reverse | 5′-GCAGTAAGTGGAGGACAGCCC-3′ | |
| NADPH oxidase-1 | Forward | 5′-GCTGGCCTTACTGGAGTGGTC-3′ |
| Reverse | 5′-TCCTGCGGATAAACTCCATAGC-3′ | |
| NADPH oxidase-4 | Forward | 5′-CTGTCCTGAACCTCAACTGCAG-3′ |
| Reverse | 5′-TGTGATCCGCGAAGGTAAGC-3′ | |
| IL-6 | Forward | 5′-CAAGAGACTTCCAGCCAGTTGC-3′ |
| Reverse | 5′-TGTTGTGGGTGGTATCCTCTGTG-3′ | |
| SOD-1 | Forward | 5′-GGGGACAATACACAAGGCTGT-3′ |
| Reverse | 5′-CCTTTCCAGCAGCCACATTG-3′ | |
| PRMT-1 | Forward | 5′-TTGACTCCTATGCCCACT -3′ |
| Reverse | 5′-CCACATCCAGCACCACC -3′ | |
| β-actin | Forward | 5′-TGTGTGGATTGGTGGCTCTATC-3′ |
| Reverse | 5′-CATCGTACTCCTGCTTGCTGATC-3′ | |
IL-6 = interleukin 6; MR = mineralocorticoid receptor; NADPH = nicotinamide adenine dinucleotide phosphate; PRMT = protein arginine N-methyltransferase; SOD = superoxide dismutase.
The following thermal cycler conditions were used: 2 min at 50°C; 10 min at 95°C; 40 cycles of 95°C for 15 s and 60°C for 1 min; 15 s at 95°C, 15 s at 60°C, and 15 s at 95°C. Primer specificity was confirmed via dissociation curve analysis after qPCR amplification.
Statistical Analyses
The results are expressed as mean ± standard error of the mean. Statistical significance was determined using 1- or 2-way analysis of variance and Bonferroni's multiple t-testing in SPSS Statistics ver.26 (IBM, NY). P-values < 0.05 were considered statistically significant.
Results
Biological Parameters
Mean body weight was similar in all 3 groups (control, 342.3 ± 7.0 g; HS, 329.3 ± 6.1 g; HS + EPL, 333.2 ± 4.8 g, P > .05). At the end of the study period, HS rats had significantly higher systolic blood pressure than the control rats (control, 125.1 ± 2.7 mmHg; HS, 225.8 ± 3.1 mmHg, P < .01), whereas HS + EPL and HS rats had similar systolic blood pressure (HS + EPL, 210.8 ± 6.3 mmHg, P > .05) (Figure 1 ).
Systolic blood pressure (SBP) at initial and final period of the study. Data are reported as mean ± standard error of the mean. ∗∗P < .01 vs each group using analysis of variance and Bonferroni's multiple. t-testing. EPL = eplerenone; HS = high salt.
Erectile Function
ICP and ICP/MAP ratios were significantly lower in HS rats than in control rats (control, 0.62 ± 0.03; HS, 0.30 ± 0.02, respectively; P < .01, respectively). HS + EPL rats had higher ICP and ICP/MAP ratio than the HS rats (0.49 ± 0.07; P < .05, respectively) (Figure 2 ).
(A) Representative recordings of intracavernosal pressure (ICP) and arterial pressure changes, during electrical stimulation of rat cavernous nerve. (B) Erectile function according to the ICP/mean arterial pressure (MAP) ratio. Data are reported as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01 vs each group using analysis of variance and Bonferroni's multiple t-testing. EPL = eplerenone; HS = high salt.
Serum ADMA and L-Arg Levels
Serum ADMA levels were significantly higher in the HS group (360.4 ± 19.8 μg/mL) than in the control (234.1 ± 13.1 μg/mL, P < .01) or HS + EPL groups (265.0 ± 38.8 μg/mL, P < .05), whereas L-Arg levels were similar in all 3 groups (control, 481.7 ± 39.8 μg/mL; HS, 474.1 ± 35.6 μg/mL; HS + EPL, 483.1 ± 49.4 μg/mL, P > .05). The L-Arg/ADMA ratio was significantly lower in the HS group than in the control and HS + EPL groups (P < .05) (Figure 3 ). Serum sodium levels did not change between the groups (control: 147.5 ± 0.65 mEq/L, HS: 149.3 ± 0.85 mEq/L, HS + EPL: 149.5 ± 0.65 mEq/L; P > .05).
Serum level of asymmetric dimethylarginine (ADMA) and L-arginine (L-Arg), and the L-Arg/ADMA ratio, in the rats. Data are reported as mean ± standard error of the mean. ∗P < .05 and ∗∗P < .01 vs each group using analysis of variance and Bonferroni's multiple t-testing. EPL = eplerenone; HS = high salt.
mRNA Expression Analysis
The MR mRNA levels in HS rats were significantly higher than those in control rats (P < .05) but significantly lower than those in HS + EPL rats (P < .05) (Figure 4 ). Expression of the inflammatory markers p22phox, NADPH oxidase-1, NADPH oxidase-4, and IL-6, and that of PRMT-1, marker of ADMA accumulation, was also significantly higher in the HS group than in the control and HS + EPL groups (P < .05). In contrast, SOD-1 expression did not differ significantly between the groups (P > .05) (Figure 5 ).
Expression of the mineralocorticoid receptor (MR) gene in rat corpus cavernosum. Target gene. expression was quantified relative to that of β-actin using the comparative CT method. Data are reported as mean ± standard error of the mean. ∗P < .05 and ∗∗P < .01 vs each group using analysis of variance and Bonferroni's multiple t-testing. EPL = eplerenone; HS = high salt.
Expression of inflammation-related and ADMA metabolism-related genes in rat corpus cavernosum. Target gene expression was quantified relative to that of β-actin using the comparative CT method. Data are reported as mean ± standard error of the mean. ∗P < .05 and ∗∗P < .01 vs each group using analysis of variance and Bonferroni's multiple t-testing. ADMA = asymmetric dimethylarginine; EPL = eplerenone; HS = high salt; IL = interleukin; NADPH = nicotinamide adenine dinucleotide phosphate; PRMT-1 = protein arginine N-methyltransferase; SOD = superoxide dismutase.
Discussion
Hypertension has been reported to cause ED in rats.22 However, our study is the first to show that high-salt diet–induced ED can occur independently of hypertension. In Sprague-Dawley rats and C57BL/6J mice, high salt intake causes endothelial dysfunction in the aorta and mesenteric artery without increasing the blood pressure.23,24 Ayuzawa and Fujita demonstrated that aberrant MR activation in the distal nephron triggers salt-induced hypertension.25 They also showed that high salt intake activates MR through Rac1, a member of the Rho family of small guanosine triphosphate-binding proteins.26 In this study, MR mRNA expression was markedly elevated in HS rats. In addition, EPL administration increased the expression because of inhibiting the MR activation.
Unlike a normal diet (0.3% NaCl), a high-salt diet (8% NaCl) increased the blood pressure of Dahl-S rats and impaired their erectile function. Daily administration of EPL prevented these adverse effects without altering the blood pressure. This is consistent with previous studies.27 Nakamura et al27 reported that EPL is protective against cardiovascular injury without affecting the blood pressure. Although hypertension is a risk factor for ED, our study indicates that high salt intake directly impairs erectile function beyond its effect on blood pressure.
ADMA is an endogenous arginine compound whose production is upregulated in certain disease states, such as hypertension, diabetes, and dyslipidemia, as a result of oxidative stress and inflammation.28 We found that serum ADMA levels were increased in HS rats, and that EPL administration attenuated this effect. ADMA reduces nitric oxide (NO) bioactivity and impairs endothelial function.29 We did not measure cavernosal ADMA concentration; however, Park et al30 reported that plasma ADMA levels reflect cavernosal ADMA accumulation. Elesber et al31 reported that elevated plasma ADMA reduces systemic NO bioactivity, leading to systemic and coronary endothelial dysfunction in patients with ED. In this study, L-Arg/ADMA ratio was decreased in the HS group; EPL treatment increased ADMA concentration, although L-Arg levels were not affected. L-Arg is a physiological substrate of NO production, and L-Arg/ADMA ratio is an indicator of endothelial function.32 Therefore, high salt intake can lead to endothelial dysfunction in the penis.
The expression of PRMT-1, a methyltransferase that catalyzes the formation of ADMA, was upregulated in the HS group. Methyltransferases are activated by oxidative stress, and ADMA production is stimulated by inflammatory cytokines in cultured endothelial cells.33 Therefore, we propose that the elevated serum ADMA levels observed in HS rats were caused by oxidative stress (as indicated by increased NADPH oxidase-1, NADPH oxidase-4, and p22phox expression) and inflammation (as indicated by increased IL-6 levels). The mRNA expressions of inflammation factors are correlated with the serum and protein levels in the CC.34 EPL administration suppressed these factors to maintain low serum ADMA levels despite the high salt intake.
To elucidate the source of inflammation in our model, we investigated the mRNA levels of various inflammatory biomarkers and antioxidants with known roles in the oxidative stress response. Our analyses revealed that HS rats upregulated the expression of proinflammatory NADPH oxidase-1, NADPH-oxidase-4, and p22phox, whereas the expression of antioxidant SOD-1 remained unchanged. NADPH oxidase-1 expressed in smooth muscle interacts with substance B to produce reactive oxygen species (ROS).35,36 NADPH oxidase-4 expressed in endothelial cells also produces ROS. Increased ROS generation leads to oxidative stress and converts NO into its inactive form, peroxynitrite (ONOO−).37 Peroxynitrite oxidizes tetrahydrobiopterine, a critical cofactor for NO synthases, which, in turn, impairs the function of endothelial NO synthase and reduces NO production.38 These reactions could be responsible for the decreased NO production in the CC of HS Dahl-S rats.
Oxidative stress and inflammation are also damaging to vascular tissue, including the CC.30,39 Moreover, oxidative stress causes DNA damage, as evidenced by elevated 8-OHdG levels in the DNA.40 We have previously shown that castrated rats display higher 8-OHdG levels than control rats, indicating that testosterone deficiency increases oxidative stress in the CC,16 which induces endothelial dysfunction.
As a limitation of this study, because this study was a pilot study and various preliminary experiments were performed, all the samples were used up, and experiments such as Western blotting or tissue levels of ADMA could not be investigated. For these reasons, we think that it needs to be shown in future experiments. A limitation of our study is also that it only targeted salt-sensitive rats. Aging, metabolic syndrome, and sympathicotonia are thought to increase salt sensitivity in humans.26,41,42 DuPont et al43 showed that endothelium-dependent dilation was impaired by high dietary sodium even in healthy salt-resistant humans. Therefore, high salt intake could induce erectile dysfunction not only in salt-sensitive but also in salt-insensitive humans. Thus, salt restriction and MR inhibitors could be beneficial as prophylactic or therapeutic agents against ED in men consuming a high-salt diet.
Conclusions
In this study, a high-salt diet enhanced MR expression and induced ED in Dahl-S rats. Selective MR inhibition improved erectile function without affecting the blood pressure. Our results suggest that high salt intake causes ED through the MR pathway beyond its effect on blood pressure. Thus, salt restriction and MR inhibitors could serve as useful prophylactic or therapeutic agents against ED in men consuming a high-salt diet.
Statement of authorship
Category 1
Conception and Design
Tomoteru Kishimoto, Masayuki Takahashi, Hiro-omi Kanayama
Acquisition of Data
Tomoya Kataoka, Yuka Yamamoto, Gakuto Asano, Ayako Fukamoto, Yuji Hotta, Yasuhiro Maeda
Analysis and Interpretation of Data
Tomoteru Kishimoto, Tomoya Kataoka, Yuka Yamamoto, Yuji Hotta, Yasuhiro Maeda, Hiro-omi Kanayama, Kazunori Kimura
Category 2
Drafting the Article
Tomoteru Kishimoto, Tomoya Kataoka, Hiro-omi Kanayama, Kazunori Kimura
Revising It for Intellectual Content
Tomoteru Kishimoto, Tomoya Kataoka, Hiro-omi Kanayama, Kazunori Kimura
Category 3
Final Approval of the Completed Article
Tomoteru Kishimoto, Tomoya Kataoka, Hiro-omi Kanayama, Kazunori Kimura
Funding:
None.
Acknowledgments
This research was partially supported by the Ministry of Education, Science, Sports, and Culture, Grant-in Aid for Challenging Exploratory Research, 2011-2012 (23659760, Tomoteru Kishimoto), The Public Foundation of Chubu Science and Technology Center, Grant-in Aid for Academic Future Research, 2017-2018 (Tomoya Kataoka), and The Salt Science Research Foundation, Grant-in Aid for Project of Medical Science, 2018 (Tomoya Kataoka).
References
Author notes
Equally contributed author
Conflict of Interest: The author reports no conflicts of interest.
