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High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats
Accepted Manuscript Title: High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats. Author: Klaudia Bielinska, Marek Radkowski, Marta Grochowska, Karol Perlejewski, Tomasz Huc, Kinga Jaworska, Daisuke Motooka, Shota Nakamura, Marcin Ufnal PII: DOI: Reference:
S0899-9007(18)30090-X https://doi.org/10.1016/j.nut.2018.03.004 NUT 10153
To appear in:
Nutrition
Received date: Accepted date:
30-8-2017 1-3-2018
Please cite this article as: Klaudia Bielinska, Marek Radkowski, Marta Grochowska, Karol Perlejewski, Tomasz Huc, Kinga Jaworska, Daisuke Motooka, Shota Nakamura, Marcin Ufnal, High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats., Nutrition (2018), https://doi.org/10.1016/j.nut.2018.03.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats.
Klaudia Bielinskaa, Marek Radkowski PhDb, Marta Grochowskab, Karol Perlejewskib, Tomasz Huca, Kinga Jaworskaa, Daisuke Motooka PhDc, Shota Nakamura PhDc, Marcin Ufnal MD, PhDa*.
a. Department of Experimental Physiology and Pathophysiology, Laboratory of Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland b. Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, Warsaw, Poland. c. Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
*
Correspondence: Marcin Ufnal, MD, PhD
Department of Experimental Physiology and Pathophysiology Medical University of Warsaw, Banacha 1B, 02-097 Warsaw, Poland Phone.: +48 22 116 6195, Fax: +48 22 116 6195 e-mail: [email protected]
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Highlights:
Increased plasma TMAO, a bacteria metabolite, is a cardiovascular risk marker.
We found that high salt intake increases plasma TMAO in rats.
High salt intake altered gut bacteria composition.
High-salt diet may affect an interplay between gut bacteria and host homeostasis.
Abstract: Background and Aims. High-salt diet is considered a cardiovascular risk factor; however, the mechanisms are not clear. Research suggests that gut bacteria-derived metabolites such as trimethylamine N-oxide (TMAO) are markers of cardiovascular diseases and may affect homeostasis in mammals. We evaluated the effect of high salt intake on gut bacteria and their metabolites plasma level. Research Methods & Procedures. 12-14-week-old Sprague Dawley rats were maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively), for two weeks. Blood plasma, urine and stools were analyzed for the concentration of TMA (TMAO-precursor), TMAO, and indoxyl sulfate, an indole metabolite. The gut-blood barrier (GBB) permeability to TMA and TMA liver clearance were assessed at baseline and after TMA intracolonic challenge test. Gut bacterial flora was analyzed with 16S rRNA gene sequence analysis. Results. Isotonic and Hypertonic groups showed a significantly higher plasma TMAO, and a significantly lower 24hr TMAO urine excretion than controls. However, TMA stool level was similar between the groups. There was no significant difference between the groups in the GBB permeability and TMA liver clearance. Plasma indoxyl concentration and 24hr urine indoxyl excretion were similar between the groups. There was a significant difference between the groups in gut bacteria composition.
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Conclusions: High salt intake increases plasma TMAO concentration, which is associated with decreased TMAO urine excretion. Furthermore, high salt intake alters gut bacteria composition. Those findings suggest that salt intake affects an interplay between gut bacteria and their host homeostasis.
Keywords: High salt intake, trimethylamine N-oxide (TMAO), gut bacteria, gut-blood barrier, cardiovascular marker
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Introduction
Rats, similarly to humans show an innate appetite for salt and consume it in the excess of their physiological needs [1]. High-salt diet is considered a risk factor for cardiovascular diseases (CVDs); however, the mechanisms whereby high salt intake promotes the development of CVDs are not clear.
Increasing evidence suggests that mammalian homeostasis strongly depends on a mutualistic relationship with gut bacteria, and that CVDs are associated with gut microbiota dysbiosis [2]. Recent clinical studies show a positive correlation between an increased plasma level of trimethylamine N-oxide (TMAO), a gut bacteria metabolite of dietary L-carnitine and choline, and an increased risk of CVDs [3, 4]. Since L-carnitine and choline are abundant in red meat, it has been proposed that TMAO may constitute a link between high red meat consumption and CVDs [5].
Indoxyl sulfate (indoxyl) is a tryptophan derived gut bacteria metabolite. Several studies show that indoxyl may affect the circulatory system functions by decreasing NO production, increasing production of reactive oxygen species, and promoting cardiac interstitial fibrosis [6, 7].
We hypothesized that high salt intake affects gut bacteria and their metabolites plasma level.
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Materials and Methods
The experiments were carried out according to Directive 2010/63/EU on the protection of animals used for scientific purposes, and were approved by the Local Bioethical Committee. All surgical procedures were performed under general anesthesia with urethane (SigmaAldrich, Poland) at a dose of 1.5 g/kg of body weight.
The study was performed on male, 12-14-week-old, Sprague Dawley rats (n=48, Central Laboratory of Experimental Animals, Centre for Preclinical Research and Technology, Warsaw, Poland). Rats were fed standard laboratory diet (0.19 % Na, Labofeed B standard, Kcynia, Poland) and were given either tap water (controls), or 0.9% NaCl water solution (Isotonic group), or 2 % NaCl water solution (Hypertonic group) for drinking for two weeks ad libitum.
Metabolic, biochemical and metagenomic analysis After two weeks of the above-mentioned treatment, controls (n=9), Isotonic (n=9), and Hypertonic (n=9) groups were maintained for 2 days in metabolism cages to evaluate 24hr water and food balance and to collect urine for TMA, TMAO, indoxyl, sodium excretion study. Samples from the second day were analyzed. Next, blood and stool samples were collected as described below.
Blood plasma tests Rats were anaesthetized and blood from the right ventricle of the heart was taken for plasma biochemical analyses. Plasma and urine sodium, potassium and creatinine were analyzed using Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, USA). Blood plasma, urine and
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stool TMA, TMAO and indoxyl concentrations were evaluated using liquid chromatography coupled with triple-quadrupole mass spectrometry, as previously described [8].
Collection and preparation of stool samples for 16S rRNA sequencing and TMA stool concentration analysis After the blood taking, rats were killed by decapitation. A 6-7 cm long segment of the colon (a middle part between the cecum and the rectum) was closed with sutures and removed. Two samples, each of 0.5 ml of stools were collected from the removed colon. The sample allocated to metagenomic analyzes was instantly frozen at -80oC. The second sample which was allocated to TMA stool concentration analyses, was weighted and homogenized with 1 ml of 0.9% NaCl in a closed 2 ml laboratory tube by vortexing it for 5 min. Afterwards, the sample was centrifuged for 5 minutes at 5,000 rpm, and 1 ml of the obtained supernatant was transferred to a laboratory tube and again centrifuged for 5 minutes. All procedures were performed at the temperature of 2-5oC. The supernatant was collected into Eppendorf tubes and frozen at -20oC.
16S rRNA metagenomics DNA was extracted from 60-80 mg of stool by Nucleospin DNA Stool Kit (Macherey-Nagel; Germany) according to manufacturer’s instructions and suspended in 100 μl of water. Amplification of the V3-V4 region of 16S rRNA gene and sequencing was performed according to 16S Metagenomic Sequencing Library Preparation protocol designed by Illumina (San Diego, USA). Sequencing was performed on Illumina MiSeq (300 nt, pairedend reads).
Gut-blood barrier permeability and liver clearance of TMA 6 Page 6 of 31
In separate experiments, controls (n=7), Isotonic (n=7) and Hypertonic (n=7) groups were implanted with polyurethane catheters inserted into the portal vein and into the inferior vena cava, just above the hepatic veins confluence. Blood samples from the veins were collected at baseline and 15 min after the intracolonic administration of TMA (1 mg/kg of body weight). TMA liver clearance was defined as the percent of TMA metabolized by the liver (1x 100%).
Statistical analyses The Kolmogorov-Smirnov test was used to test normality of the distribution. Differences between the groups were evaluated by one-way ANOVA, followed by Tukey’s post hoc test or T-test were apropriate. A value of two-sided p<0.05 was considered significant. Analyses were conducted using Dell Statistica, version 13 (Dell Inc, Tulsa, USA). Diversity analyses and operational taxonomic units (OTUs) picking in 16S rRNA metagenomics were performed using an open-reference OTU picking protocol by searching reads against the Greengenes database (97% similarity threshold) following QIIME workflow for Illumina NGS data [9]. The relationships between the community structures of the gut microbiota were examined using the Principal Coordinate Analysis (PCoA) based on the unweighted Unifrac distance matrixes. Taxonomic differences were compared across groups to determine if particular genera were associated with the treatment. For this purpose, Kruskal-Wallis test with FDR multiple test correction was performed.
Results
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Metabolic, biochemical and metagenomic analysis
General metabolic and biochemical parameters Controls and the Isotonic group showed a similar food ingestion and body weight gain during the experiments, whereas the Hypertonic group showed decreased food ingestion and body weight. There was no significand difference in plasma sodium between controls and the Isotonic group, whereas the Hypertonic group showed an increased plasma sodium level. There was no significant difference between controls and Isotonic and Hypertonic groups in plasma potassium and creatinine level. Hypertonic and Isotonic groups showed a significantly higher fluid intake, urine output, and 24hr urine sodium excretion than controls (Table 1).
Bacteria metabolites in stools, plasma and urine There was no significant difference between the groups in stool density, TMA stool level, and TMA plasma level (Table 1, Fig. 1). In contrast, Isotonic and Hypertonic groups showed significantly higher plasma TMAO level (F2,19=3.86, p<0.05) and significantly lower 24hr TMAO urine excretion (F2,19=6.6, p<0.05) in comparison to controls. There was no significant difference in plasma indoxyl level and 24hr urine indoxyl excretion between the groups (Fig. 1).
16S rRNA metagenomics 8 Page 8 of 31
All samples were sequenced in the same run yielding a total of 3,734,557 reads. The number of reads ranged from 83,480 to 206,507 resulting in an average 138,317 reads per sample. After quality processing one control sample was excluded from further analyses (controls n=8, Isotonic n=9, and Hypertonic n=9 groups).
Alpha diversity analyses, describing species richness, showed no significant differences between the treatment groups based on the presence of observed OUTs in samples (Fig. 2). Shannon diversity index (H) was similar between samples (mean: controls=6.85; the Isotonic group=7.23; the Hypertonic group=7.42).
Beta diversity analysis, showing the relationships between the community structures of the gut microbiota, was examined using PCoA based on the unweighted Unifrac distance matrixes. PCoA showed a significant difference between the groups (ANOSIM p=0.001), (Fig. 3).
We found a significant difference between the groups in 21 bacterial genera including: Akkermansia, SMB53, Proteus, Facklamia, Corynebacterium, Sarcina, Lachnospira, Staphylococcus, 02d06, Aggregatibacter, Actinomyces, Helicobacter, Turicibacter, Streptococcus, Acinetobacter, Rothia, Aerococcus, Anaerovibrio, Candidatus Arthromitus, Prevotella, Jeotgalicoccus, (Table 2).
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Out of identified bacterial genera we found five bacterial genera with TMA producing capacity[10, 11] i.e. Clostridium, Collinsella, Desulfovibrio, Lactobacillus, Proteus. However, there was no significant difference between the groups in the genera composition (Fig. 4).
Gut-blood barrier permeability and liver clearance of TMA
Gut-blood barrier permeability At baseline, there was no significant difference between the groups in TMA portal plasma level. Intracolonic administration of TMA produced a significant increase in portal blood TMA. The size of the increase was similar between the groups (Fig. 5A).
Liver clearance of TMA TMA liver clearance was similar between the groups at baseline, and 15 min after the intracolonic administration of TMA (Fig. 5B).
Discussion
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A new finding of our study is that high salt intake is associated with increased plasma TMAO, and reduced TMAO urine excretion. Furthermore, we found that high sodium intake affects gut microbiota composition.
High-salt diet is a risk factor for CVDs, however, significant individual differences between humans and rats in salt-sensitivity have been shown[12]. Interestingly, several clinical trials show a positive correlation between plasma TMAO, a gut bacteria metabolite, and cardiovascular risk[3, 13]. Plasma TMAO increases after ingesting choline and L-carnitine that are abundant in red meat[14]. It has also been suggested that high plasma TMAO is associated with dairy consumption[15], however, other studies did not confirmed this relationship [16]. Intriguingly, several-fold higher TMAO plasma concentrations were found after fish intake in comparison to intake of red meat or eggs[17]. Likewise, Cheung et al. showed, that TMAO is the best biomarker of fish intake, when compared to a diet of different composition[18].
In this study, we found that rats maintained on 0.9% and 2% NaCl water solution had significantly higher plasma TMAO than controls maintained on tap water. This suggests that high salt intake is another factor that may increase plasma TMAO concentration.
Plasma TMAO originates form trimethylamine (TMA), a product of gut bacteria metabolism. TMA is absorbed from the large bowel and is carried by portal blood to the liver. In the liver the majority of TMA is oxidized to TMAO by flavin-containing monooxygenase-3 (FMO3). TMA and TMAO is excreted mainly with the urine. Therefore, besides diet, blood TMAO concentration depends on microbiota activity (production of TMA), the permeability of the
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gut-blood barrier (GBB) to TMA, the oxidation of TMA by the liver, and the excretion of TMA and TMAO (reviewed [14]).
To evaluate the effect of high salt intake on the gut bacteria production of TMA, we checked TMA stool concentration and gut bacteria composition with regard to bacterial genera such as Clostridium, Collinsella, Desulfovibrio, Lactobacillus, Proteus, which are thought to produce methylamines including TMA [10, 11]. We found differences, however not significant, in Lactobacillus genus between controls and rats on high salt intake. Nevertheless, the stool TMA level was comparable between the groups, suggesting the lack of a significant effect of high salt intake on gut bacteria TMA production.
Importantly, we found a significant effect of high salt intake on the overall composition of gut bacteria in rats. This may not be surprising, as salt has been used as an antiseptic and a food preservative, inhibiting bacterial proliferation by producing hyperosmotic environment. In fact, the ability of bacteria to adapt to changes in external osmolarity directly determines the survival and proliferation of bacteria[19]. Since the salt tolerance differs between bacterial strains[20], high salt intake may favor proliferation of some bacterial strains over others. Changes in the gut bacteria composition in mice on high salt intake has been just recently reported in unpublished conference data by others [21].
Gut bacteria metabolites may affect mammalian homeostasis entering the circulation. The access of gut-derived molecules such as TMA to the bloodstream is guarded by the GBB. It has been suggested that functions of the GBB is disturbed in CVDs [22-24]. Some studies suggest that physiological gut microbiota plays an important role in maintaining the GBB
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integrity[25]. In the present study, we found that high salt intake does not affect the GBB permeability to TMA, despite the associated gut dysbiosis.
Another factor affecting plasma TMAO concentration is the oxidation of TMA to TMAO by the liver FMO3. Mutations in the FMO3 gene have been shown to produce trimethylaminuria, or fish-odor syndrome, the name which comes from the characteristic odor of decaying fish for which TMA is responsible[26]. In this study, we found no effect of high salt intake on TMA liver clearance.
Our findings suggest that the most likely mechanism responsible for increased plasma TMAO concentration in Isosmotic and Hyperosmotic groups was decreased excretion of TMAO with urine. Namely, we found that high salt intake increased blood TMAO level, whereas decreased 24hr TMAO urine excretion. This suggests that plasma TMAO accumulation is due to the retention of TMAO by the kidneys.
On the one hand, the retention of TMAO in rats receiving salt overload may reflect some pathological changes associated with water-electrolyte balance disturbances. Kidneys play a key role in maintaining homeostasis by regulating water-electrolyte balance and removing waste products. In our study, controls and the Isotonic group maintained normal plasma sodium level. In contrast, in the Hypertonic group the excessive sodium intake exceeded the kidney capacity to maintain normal plasma sodium level, despite a significant increase in sodium urine excretion. We found increased plasma TMAO and decreased TMAO urine excretion in both, the Isotonic and the Hypertonic group. Therefore, it seems that an elevated plasma TMAO was triggered by high salt intake rather than high plasma sodium. There is some evidence, that plasma TMAO increases in kidney failure [27]. However, in our study we
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found no evident markers of kidney failure in Isosmotic and Hyperosmotic groups with regard to removing waste products. Namely, plasma creatinine and potassium levels were comparable between the groups. Furthermore, the excretion of TMA, a TMAO precursor, as well as excretion of indoxyl were similar between controls and rats on high salt intake.
On the other hand, it is possible that the accumulation of TMAO may be an adaptation mechanism that prepares an organism to an osmotic stress caused by high salt intake. Interestingly, it has been shown that TMAO is utilized by numerous animals as an osmolyte. Osmolytes are used by cells to maintain cell volume despite osmotic and hydrostatic stresses [28]. For example, TMAO is used by saltwater fish to protect their cells from osmotic stress in the hyperosmotic environment [28]. Furthermore, TMAO has been shown to neutralize perturbations of protein structure and function caused by changes in osmolarity, hydrostatic pressure and urea [29]. Research suggests that methylamines are accumulated in mammalian kidney medulla in which osmolarity may be four times higher than osmolarity of plasma [30].
In the present study, we did not find a significant effect of high salt intake on plasma level and urine excretion of indoxyl sulfate, suggesting no significant impact of high salt intake on indole and its derivatives balance in rats.
In conclusion, our study shows that high salt intake in rats is associated with increased plasma TMAO level. Whether the TMAO accumulation has a harmful, beneficial or no significant effect on animals consuming the excess of salt requires further investigation. Nevertheless, our study suggests that high-salt diet may be a confounding factor in clinical studies evaluating plasma TMAO level. Finally, we showed that high salt intake produces gut
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dysbiosis. Taking together, our findings suggest that salt intake may affect an interplay between the gut bacteria and the host homeostasis.
Acknowledgements This work was supported by National Science Centre, Poland grant no. 2016/21/B/NZ5/02544. Conception and design of the work - MU. Acquisition, analysis, and interpretation of data for the work; KB, MR, MG, KP, TH, KJ, DM, SN, MU. Drafting the work – MU, KB, MR, KP, KB, DM, SN.
Conflicts of interest None.
References
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[7] Yisireyili M, Shimizu H, Saito S, Enomoto A, Nishijima F, Niwa T. Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats. Life sciences. 2013;92:1180-5. [8] Ufnal M, Jazwiec R, Dadlez M, Drapala A, Sikora M, Skrzypecki J. Trimethylamine-N-oxide: a carnitine-derived metabolite that prolongs the hypertensive effect of angiotensin II in rats. Can J Cardiol. 2014;30:1700-5. [9] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335-6. [10] Craciun S, Balskus EP. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:21307-12. [11] Romano KA, Vivas EI, Amador-Noguez D, Rey FE. Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the proatherogenic metabolite trimethylamine-N-oxide. mBio. 2015;6:e02481. [12] Sanders PW. Dietary salt intake, salt sensitivity, and cardiovascular health. Hypertension. 2009;53:442-5. [13] Senthong V, Wang Z, Fan Y, Wu Y, Hazen SL, Tang WH. Trimethylamine N-Oxide and Mortality Risk in Patients With Peripheral Artery Disease. Journal of the American Heart Association. 2016;5. [14] Ufnal M, Zadlo A, Ostaszewski R. TMAO: A small molecule of great expectations. Nutrition. 2015;31:1317-23. [15] Rohrmann S, Linseisen J, Allenspach M, von Eckardstein A, Muller D. Plasma Concentrations of Trimethylamine-N-oxide Are Directly Associated with Dairy Food Consumption and Low-Grade Inflammation in a German Adult Population. The Journal of nutrition. 2016;146:283-9. [16] Obeid R, Awwad HM, Keller M, Geisel J. Trimethylamine-N-oxide and its biological variations in vegetarians. European journal of nutrition. 2016. [17] Cho CE, Taesuwan S, Malysheva OV, Bender E, Tulchinsky NF, Yan J, et al. Trimethylamine-N-oxide (TMAO) response to animal source foods varies among healthy young men and is influenced by their gut microbiota composition: A randomized controlled trial. Molecular nutrition & food research. 2017;61. [18] Cheung W, Keski-Rahkonen P, Assi N, Ferrari P, Freisling H, Rinaldi S, et al. A metabolomic study of biomarkers of meat and fish intake. The American journal of clinical nutrition. 2017;105:600-8. [19] Sleator RD, Hill C. Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS microbiology reviews. 2002;26:49-71. [20] Marshall BJ, Ohye DF, Christian JH. Tolerance of bacteria to high concentrations of NaCl and glycerol in the growth medium. Applied microbiology. 1971;21:363-4. [21] Hung S-C, Yang T-M, Tarng D-C. SP260HIGH SALT DIET ALTERS GUT MICROBIOTA LEADING TO INFLAMMATION AND PROGRESSION OF CKD. Nephrology Dialysis Transplantation. 2017;32:iii194-iii. [22] Sandek A, Bauditz J, Swidsinski A, Buhner S, Weber-Eibel J, von Haehling S, et al. Altered intestinal function in patients with chronic heart failure. Journal of the American College of Cardiology. 2007;50:1561-9. [23] Santisteban MM, Qi Y, Zubcevic J, Kim S, Yang T, Shenoy V, et al. Hypertension-Linked Pathophysiological Alterations in the Gut. Circulation research. 2017;120:312-23.
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[24] Ufnal M, Pham K. The gut-blood barrier permeability - A new marker in cardiovascular and metabolic diseases? Med Hypotheses. 2017;98:35-7. [25] Camilleri M, Madsen K, Spiller R, Greenwood-Van Meerveld B, Verne GN. Intestinal barrier function in health and gastrointestinal disease. Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society. 2012;24:503-12. [26] Treacy EP, Akerman BR, Chow LM, Youil R, Bibeau C, Lin J, et al. Mutations of the flavincontaining monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Human molecular genetics. 1998;7:839-45. [27] Tang WH, Wang Z, Kennedy DJ, Wu Y, Buffa JA, Agatisa-Boyle B, et al. Gut microbiotadependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease. Circulation research. 2015;116:448-55. [28] Yancey PH, Siebenaller JF. Co-evolution of proteins and solutions: protein adaptation versus cytoprotective micromolecules and their roles in marine organisms. The Journal of experimental biology. 2015;218:1880-96. [29] Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. The Journal of experimental biology. 2005;208:2819-30. [30] Somero G. From Dogfish to Dogs: Trimethylamines Protect Proteins from Urea. Physiology. 1986;1:9-12.
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Figure 1. (A) stool concentration, (B) peripheral blood plasma concentration, and (C) 24hr urine excretion in in rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively). Trimethylamine (TMA), trimethylamine N-oxide (TMAO) and indoxyl sulfate (Indoxyl). Values are means, ± SE. ANOVA followed by post-hoc Tuckey-test (* - p < 0.05).
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Figure 2. Alpha diversity analysis: A) Similar number of observed operational taxonomic units (OTUs) in stools of rats maintained on tap water (controls), rats maintained on 0.9% NaCl solution (Isotonic group) and rats maintained on 2% NaCl solution (Hypertonic group) for two weeks. B) No significant differences in species richness among analyzed groups based on Shannon diversity index.
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Figure 3. Principal coordinate analysis of 16S sequences from 26 stool samples using unweighted UniFrac. Rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively).
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Figure 4. The percentage of reads mapped to bacterial genera with TMA producing capacity (Clostridium, Collinsella, Desulfovibrio, Lactobacillus, Proteus). Stool samples form rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively).
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Figure 5. (A) Trimethylamine (TMA) portal blood level (µg/ml) at baseline (0) and 15 min after the intracolonic administration of TMA (1 mg/kg of body weight), (IC TMA). (B) TMA liver clearance at baseline and 15 min after the intracolonic administration of TMA. TMA liver clearance is defined as the percent of TMA metabolized by the liver (1x 100%). Rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively). Values are means, + SE. * - p<0.05 vs baseline (paired T-Test).
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Table 1. Body weight, food intake and water-electrolyte balance in rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively). Values are means, ± SE. ANOVA followed by post-hoc Tuckey-test. Electrolytes measured only in haemolysis-free plasma samples. † - Controls vs Hypertonic group, ‡ - Isotonic group vs Hypertonic group. Group/
Controls
Isotonic
Hypertonic
ANOVA
358.9±5.0
354.1±3.9
352.1±3.5
ns
409.3±5.9
402.7±5.0
344.4±10.2
(F2,24=23.0,
Parameter Body mass (g), the first day of the experiment Body mass (g), the last
p<0.05) †,‡
day of the experiment Change in body mass (g)
50.3±2.9
48.6±4.8
-7.7±7.8
(F2,24=35.6, p<0.05) †,‡
24hr food intake (g)
28.1±0.8
28.9±0.9
19.4±1.2
(F2,24=25.4, p<0.05) †,‡
24hr water intake (ml)
29.1±1.1
49.8±3.2
139.5±19.7
(F2,24=25.6, p<0.05) †,‡
24hr urine output (ml)
12.2±1.2
22.4±2.4
105.9±17.4
(F2,24=25.5, p<0.05) †,‡
24hr stool output (g)
15.4±0.7
15.7±1.6
9.3±0.8
(F2,24=10.7, p<0.05) †,‡
Stool density (g/ml)
1.14±0.03
1.18±0.04
1.12±0.03
ns
Plasma sodium (g/ml)
3.091±0.016
3.085±0.013
3.364±0.101
(F2,19=7.1, p<0.05)†,‡
Plasma potassium (g/ml)
0.175±0.006
0.174±0.005
0.172±0.012
ns 23 Page 23 of 31
Plasma creatinine (g/ml)
0.0086±0.0006
0.0083±0.0004 0.0076±0.0007 ns
24hr sodium urine
0.043±0.005
0.191±0.016
excretion (g)
1.087±0.227
(F2,24=20.3, p<0.05) †,‡
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Table 2 . Analysis of bacterial genera in stools of rats maintained on tap water (controls), rats maintained on 0.9% NaCl solution (Isotonic group) and rats maintained on 2% NaCl solution (Hypertonic group) for two weeks. The table shows significant differences between controls, Isotonic and Hypertonic groups in bacterial genera, p < 0.05, by Kruskal-Wallis test with FDR multiple test correction. Genus FDR p value Akkermansia
0.006
SMB53
0.006
Proteus
0.006
Facklamia
0.006
Corynebacterium
0.007
Sarcina
0.008
Lachnospira
0.011
Staphylococcus
0.012
02d06
0.016
Aggregatibacter
0.018
Actinomyces
0.018
Helicobacter
0.019
Turicibacter
0.020
Streptococcus
0.023
Acinetobacter
0.027
Rothia
0.030
Aerococcus
0.030
Anaerovibrio
0.034
Candidatus 0.044 Arthromitus Prevotella
0.044
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Jeotgalicoccus
0.044
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Figure 3_color_300dpi.tif
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Figure 4_dopasowana_300dpi.tif
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Figure_1_metabolites.tif
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Figure_2_dopasowana_300dpi.jpg
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Figure_5_bariera_klirens.tif
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S0899-9007(18)30090-X https://doi.org/10.1016/j.nut.2018.03.004 NUT 10153
To appear in:
Nutrition
Received date: Accepted date:
30-8-2017 1-3-2018
Please cite this article as: Klaudia Bielinska, Marek Radkowski, Marta Grochowska, Karol Perlejewski, Tomasz Huc, Kinga Jaworska, Daisuke Motooka, Shota Nakamura, Marcin Ufnal, High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats., Nutrition (2018), https://doi.org/10.1016/j.nut.2018.03.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats.
Klaudia Bielinskaa, Marek Radkowski PhDb, Marta Grochowskab, Karol Perlejewskib, Tomasz Huca, Kinga Jaworskaa, Daisuke Motooka PhDc, Shota Nakamura PhDc, Marcin Ufnal MD, PhDa*.
a. Department of Experimental Physiology and Pathophysiology, Laboratory of Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland b. Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, Warsaw, Poland. c. Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
*
Correspondence: Marcin Ufnal, MD, PhD
Department of Experimental Physiology and Pathophysiology Medical University of Warsaw, Banacha 1B, 02-097 Warsaw, Poland Phone.: +48 22 116 6195, Fax: +48 22 116 6195 e-mail: [email protected]
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Highlights:
Increased plasma TMAO, a bacteria metabolite, is a cardiovascular risk marker.
We found that high salt intake increases plasma TMAO in rats.
High salt intake altered gut bacteria composition.
High-salt diet may affect an interplay between gut bacteria and host homeostasis.
Abstract: Background and Aims. High-salt diet is considered a cardiovascular risk factor; however, the mechanisms are not clear. Research suggests that gut bacteria-derived metabolites such as trimethylamine N-oxide (TMAO) are markers of cardiovascular diseases and may affect homeostasis in mammals. We evaluated the effect of high salt intake on gut bacteria and their metabolites plasma level. Research Methods & Procedures. 12-14-week-old Sprague Dawley rats were maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively), for two weeks. Blood plasma, urine and stools were analyzed for the concentration of TMA (TMAO-precursor), TMAO, and indoxyl sulfate, an indole metabolite. The gut-blood barrier (GBB) permeability to TMA and TMA liver clearance were assessed at baseline and after TMA intracolonic challenge test. Gut bacterial flora was analyzed with 16S rRNA gene sequence analysis. Results. Isotonic and Hypertonic groups showed a significantly higher plasma TMAO, and a significantly lower 24hr TMAO urine excretion than controls. However, TMA stool level was similar between the groups. There was no significant difference between the groups in the GBB permeability and TMA liver clearance. Plasma indoxyl concentration and 24hr urine indoxyl excretion were similar between the groups. There was a significant difference between the groups in gut bacteria composition.
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Conclusions: High salt intake increases plasma TMAO concentration, which is associated with decreased TMAO urine excretion. Furthermore, high salt intake alters gut bacteria composition. Those findings suggest that salt intake affects an interplay between gut bacteria and their host homeostasis.
Keywords: High salt intake, trimethylamine N-oxide (TMAO), gut bacteria, gut-blood barrier, cardiovascular marker
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Introduction
Rats, similarly to humans show an innate appetite for salt and consume it in the excess of their physiological needs [1]. High-salt diet is considered a risk factor for cardiovascular diseases (CVDs); however, the mechanisms whereby high salt intake promotes the development of CVDs are not clear.
Increasing evidence suggests that mammalian homeostasis strongly depends on a mutualistic relationship with gut bacteria, and that CVDs are associated with gut microbiota dysbiosis [2]. Recent clinical studies show a positive correlation between an increased plasma level of trimethylamine N-oxide (TMAO), a gut bacteria metabolite of dietary L-carnitine and choline, and an increased risk of CVDs [3, 4]. Since L-carnitine and choline are abundant in red meat, it has been proposed that TMAO may constitute a link between high red meat consumption and CVDs [5].
Indoxyl sulfate (indoxyl) is a tryptophan derived gut bacteria metabolite. Several studies show that indoxyl may affect the circulatory system functions by decreasing NO production, increasing production of reactive oxygen species, and promoting cardiac interstitial fibrosis [6, 7].
We hypothesized that high salt intake affects gut bacteria and their metabolites plasma level.
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Materials and Methods
The experiments were carried out according to Directive 2010/63/EU on the protection of animals used for scientific purposes, and were approved by the Local Bioethical Committee. All surgical procedures were performed under general anesthesia with urethane (SigmaAldrich, Poland) at a dose of 1.5 g/kg of body weight.
The study was performed on male, 12-14-week-old, Sprague Dawley rats (n=48, Central Laboratory of Experimental Animals, Centre for Preclinical Research and Technology, Warsaw, Poland). Rats were fed standard laboratory diet (0.19 % Na, Labofeed B standard, Kcynia, Poland) and were given either tap water (controls), or 0.9% NaCl water solution (Isotonic group), or 2 % NaCl water solution (Hypertonic group) for drinking for two weeks ad libitum.
Metabolic, biochemical and metagenomic analysis After two weeks of the above-mentioned treatment, controls (n=9), Isotonic (n=9), and Hypertonic (n=9) groups were maintained for 2 days in metabolism cages to evaluate 24hr water and food balance and to collect urine for TMA, TMAO, indoxyl, sodium excretion study. Samples from the second day were analyzed. Next, blood and stool samples were collected as described below.
Blood plasma tests Rats were anaesthetized and blood from the right ventricle of the heart was taken for plasma biochemical analyses. Plasma and urine sodium, potassium and creatinine were analyzed using Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, USA). Blood plasma, urine and
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stool TMA, TMAO and indoxyl concentrations were evaluated using liquid chromatography coupled with triple-quadrupole mass spectrometry, as previously described [8].
Collection and preparation of stool samples for 16S rRNA sequencing and TMA stool concentration analysis After the blood taking, rats were killed by decapitation. A 6-7 cm long segment of the colon (a middle part between the cecum and the rectum) was closed with sutures and removed. Two samples, each of 0.5 ml of stools were collected from the removed colon. The sample allocated to metagenomic analyzes was instantly frozen at -80oC. The second sample which was allocated to TMA stool concentration analyses, was weighted and homogenized with 1 ml of 0.9% NaCl in a closed 2 ml laboratory tube by vortexing it for 5 min. Afterwards, the sample was centrifuged for 5 minutes at 5,000 rpm, and 1 ml of the obtained supernatant was transferred to a laboratory tube and again centrifuged for 5 minutes. All procedures were performed at the temperature of 2-5oC. The supernatant was collected into Eppendorf tubes and frozen at -20oC.
16S rRNA metagenomics DNA was extracted from 60-80 mg of stool by Nucleospin DNA Stool Kit (Macherey-Nagel; Germany) according to manufacturer’s instructions and suspended in 100 μl of water. Amplification of the V3-V4 region of 16S rRNA gene and sequencing was performed according to 16S Metagenomic Sequencing Library Preparation protocol designed by Illumina (San Diego, USA). Sequencing was performed on Illumina MiSeq (300 nt, pairedend reads).
Gut-blood barrier permeability and liver clearance of TMA 6 Page 6 of 31
In separate experiments, controls (n=7), Isotonic (n=7) and Hypertonic (n=7) groups were implanted with polyurethane catheters inserted into the portal vein and into the inferior vena cava, just above the hepatic veins confluence. Blood samples from the veins were collected at baseline and 15 min after the intracolonic administration of TMA (1 mg/kg of body weight). TMA liver clearance was defined as the percent of TMA metabolized by the liver (1x 100%).
Statistical analyses The Kolmogorov-Smirnov test was used to test normality of the distribution. Differences between the groups were evaluated by one-way ANOVA, followed by Tukey’s post hoc test or T-test were apropriate. A value of two-sided p<0.05 was considered significant. Analyses were conducted using Dell Statistica, version 13 (Dell Inc, Tulsa, USA). Diversity analyses and operational taxonomic units (OTUs) picking in 16S rRNA metagenomics were performed using an open-reference OTU picking protocol by searching reads against the Greengenes database (97% similarity threshold) following QIIME workflow for Illumina NGS data [9]. The relationships between the community structures of the gut microbiota were examined using the Principal Coordinate Analysis (PCoA) based on the unweighted Unifrac distance matrixes. Taxonomic differences were compared across groups to determine if particular genera were associated with the treatment. For this purpose, Kruskal-Wallis test with FDR multiple test correction was performed.
Results
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Metabolic, biochemical and metagenomic analysis
General metabolic and biochemical parameters Controls and the Isotonic group showed a similar food ingestion and body weight gain during the experiments, whereas the Hypertonic group showed decreased food ingestion and body weight. There was no significand difference in plasma sodium between controls and the Isotonic group, whereas the Hypertonic group showed an increased plasma sodium level. There was no significant difference between controls and Isotonic and Hypertonic groups in plasma potassium and creatinine level. Hypertonic and Isotonic groups showed a significantly higher fluid intake, urine output, and 24hr urine sodium excretion than controls (Table 1).
Bacteria metabolites in stools, plasma and urine There was no significant difference between the groups in stool density, TMA stool level, and TMA plasma level (Table 1, Fig. 1). In contrast, Isotonic and Hypertonic groups showed significantly higher plasma TMAO level (F2,19=3.86, p<0.05) and significantly lower 24hr TMAO urine excretion (F2,19=6.6, p<0.05) in comparison to controls. There was no significant difference in plasma indoxyl level and 24hr urine indoxyl excretion between the groups (Fig. 1).
16S rRNA metagenomics 8 Page 8 of 31
All samples were sequenced in the same run yielding a total of 3,734,557 reads. The number of reads ranged from 83,480 to 206,507 resulting in an average 138,317 reads per sample. After quality processing one control sample was excluded from further analyses (controls n=8, Isotonic n=9, and Hypertonic n=9 groups).
Alpha diversity analyses, describing species richness, showed no significant differences between the treatment groups based on the presence of observed OUTs in samples (Fig. 2). Shannon diversity index (H) was similar between samples (mean: controls=6.85; the Isotonic group=7.23; the Hypertonic group=7.42).
Beta diversity analysis, showing the relationships between the community structures of the gut microbiota, was examined using PCoA based on the unweighted Unifrac distance matrixes. PCoA showed a significant difference between the groups (ANOSIM p=0.001), (Fig. 3).
We found a significant difference between the groups in 21 bacterial genera including: Akkermansia, SMB53, Proteus, Facklamia, Corynebacterium, Sarcina, Lachnospira, Staphylococcus, 02d06, Aggregatibacter, Actinomyces, Helicobacter, Turicibacter, Streptococcus, Acinetobacter, Rothia, Aerococcus, Anaerovibrio, Candidatus Arthromitus, Prevotella, Jeotgalicoccus, (Table 2).
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Out of identified bacterial genera we found five bacterial genera with TMA producing capacity[10, 11] i.e. Clostridium, Collinsella, Desulfovibrio, Lactobacillus, Proteus. However, there was no significant difference between the groups in the genera composition (Fig. 4).
Gut-blood barrier permeability and liver clearance of TMA
Gut-blood barrier permeability At baseline, there was no significant difference between the groups in TMA portal plasma level. Intracolonic administration of TMA produced a significant increase in portal blood TMA. The size of the increase was similar between the groups (Fig. 5A).
Liver clearance of TMA TMA liver clearance was similar between the groups at baseline, and 15 min after the intracolonic administration of TMA (Fig. 5B).
Discussion
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A new finding of our study is that high salt intake is associated with increased plasma TMAO, and reduced TMAO urine excretion. Furthermore, we found that high sodium intake affects gut microbiota composition.
High-salt diet is a risk factor for CVDs, however, significant individual differences between humans and rats in salt-sensitivity have been shown[12]. Interestingly, several clinical trials show a positive correlation between plasma TMAO, a gut bacteria metabolite, and cardiovascular risk[3, 13]. Plasma TMAO increases after ingesting choline and L-carnitine that are abundant in red meat[14]. It has also been suggested that high plasma TMAO is associated with dairy consumption[15], however, other studies did not confirmed this relationship [16]. Intriguingly, several-fold higher TMAO plasma concentrations were found after fish intake in comparison to intake of red meat or eggs[17]. Likewise, Cheung et al. showed, that TMAO is the best biomarker of fish intake, when compared to a diet of different composition[18].
In this study, we found that rats maintained on 0.9% and 2% NaCl water solution had significantly higher plasma TMAO than controls maintained on tap water. This suggests that high salt intake is another factor that may increase plasma TMAO concentration.
Plasma TMAO originates form trimethylamine (TMA), a product of gut bacteria metabolism. TMA is absorbed from the large bowel and is carried by portal blood to the liver. In the liver the majority of TMA is oxidized to TMAO by flavin-containing monooxygenase-3 (FMO3). TMA and TMAO is excreted mainly with the urine. Therefore, besides diet, blood TMAO concentration depends on microbiota activity (production of TMA), the permeability of the
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gut-blood barrier (GBB) to TMA, the oxidation of TMA by the liver, and the excretion of TMA and TMAO (reviewed [14]).
To evaluate the effect of high salt intake on the gut bacteria production of TMA, we checked TMA stool concentration and gut bacteria composition with regard to bacterial genera such as Clostridium, Collinsella, Desulfovibrio, Lactobacillus, Proteus, which are thought to produce methylamines including TMA [10, 11]. We found differences, however not significant, in Lactobacillus genus between controls and rats on high salt intake. Nevertheless, the stool TMA level was comparable between the groups, suggesting the lack of a significant effect of high salt intake on gut bacteria TMA production.
Importantly, we found a significant effect of high salt intake on the overall composition of gut bacteria in rats. This may not be surprising, as salt has been used as an antiseptic and a food preservative, inhibiting bacterial proliferation by producing hyperosmotic environment. In fact, the ability of bacteria to adapt to changes in external osmolarity directly determines the survival and proliferation of bacteria[19]. Since the salt tolerance differs between bacterial strains[20], high salt intake may favor proliferation of some bacterial strains over others. Changes in the gut bacteria composition in mice on high salt intake has been just recently reported in unpublished conference data by others [21].
Gut bacteria metabolites may affect mammalian homeostasis entering the circulation. The access of gut-derived molecules such as TMA to the bloodstream is guarded by the GBB. It has been suggested that functions of the GBB is disturbed in CVDs [22-24]. Some studies suggest that physiological gut microbiota plays an important role in maintaining the GBB
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integrity[25]. In the present study, we found that high salt intake does not affect the GBB permeability to TMA, despite the associated gut dysbiosis.
Another factor affecting plasma TMAO concentration is the oxidation of TMA to TMAO by the liver FMO3. Mutations in the FMO3 gene have been shown to produce trimethylaminuria, or fish-odor syndrome, the name which comes from the characteristic odor of decaying fish for which TMA is responsible[26]. In this study, we found no effect of high salt intake on TMA liver clearance.
Our findings suggest that the most likely mechanism responsible for increased plasma TMAO concentration in Isosmotic and Hyperosmotic groups was decreased excretion of TMAO with urine. Namely, we found that high salt intake increased blood TMAO level, whereas decreased 24hr TMAO urine excretion. This suggests that plasma TMAO accumulation is due to the retention of TMAO by the kidneys.
On the one hand, the retention of TMAO in rats receiving salt overload may reflect some pathological changes associated with water-electrolyte balance disturbances. Kidneys play a key role in maintaining homeostasis by regulating water-electrolyte balance and removing waste products. In our study, controls and the Isotonic group maintained normal plasma sodium level. In contrast, in the Hypertonic group the excessive sodium intake exceeded the kidney capacity to maintain normal plasma sodium level, despite a significant increase in sodium urine excretion. We found increased plasma TMAO and decreased TMAO urine excretion in both, the Isotonic and the Hypertonic group. Therefore, it seems that an elevated plasma TMAO was triggered by high salt intake rather than high plasma sodium. There is some evidence, that plasma TMAO increases in kidney failure [27]. However, in our study we
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found no evident markers of kidney failure in Isosmotic and Hyperosmotic groups with regard to removing waste products. Namely, plasma creatinine and potassium levels were comparable between the groups. Furthermore, the excretion of TMA, a TMAO precursor, as well as excretion of indoxyl were similar between controls and rats on high salt intake.
On the other hand, it is possible that the accumulation of TMAO may be an adaptation mechanism that prepares an organism to an osmotic stress caused by high salt intake. Interestingly, it has been shown that TMAO is utilized by numerous animals as an osmolyte. Osmolytes are used by cells to maintain cell volume despite osmotic and hydrostatic stresses [28]. For example, TMAO is used by saltwater fish to protect their cells from osmotic stress in the hyperosmotic environment [28]. Furthermore, TMAO has been shown to neutralize perturbations of protein structure and function caused by changes in osmolarity, hydrostatic pressure and urea [29]. Research suggests that methylamines are accumulated in mammalian kidney medulla in which osmolarity may be four times higher than osmolarity of plasma [30].
In the present study, we did not find a significant effect of high salt intake on plasma level and urine excretion of indoxyl sulfate, suggesting no significant impact of high salt intake on indole and its derivatives balance in rats.
In conclusion, our study shows that high salt intake in rats is associated with increased plasma TMAO level. Whether the TMAO accumulation has a harmful, beneficial or no significant effect on animals consuming the excess of salt requires further investigation. Nevertheless, our study suggests that high-salt diet may be a confounding factor in clinical studies evaluating plasma TMAO level. Finally, we showed that high salt intake produces gut
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dysbiosis. Taking together, our findings suggest that salt intake may affect an interplay between the gut bacteria and the host homeostasis.
Acknowledgements This work was supported by National Science Centre, Poland grant no. 2016/21/B/NZ5/02544. Conception and design of the work - MU. Acquisition, analysis, and interpretation of data for the work; KB, MR, MG, KP, TH, KJ, DM, SN, MU. Drafting the work – MU, KB, MR, KP, KB, DM, SN.
Conflicts of interest None.
References
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[7] Yisireyili M, Shimizu H, Saito S, Enomoto A, Nishijima F, Niwa T. Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats. Life sciences. 2013;92:1180-5. [8] Ufnal M, Jazwiec R, Dadlez M, Drapala A, Sikora M, Skrzypecki J. Trimethylamine-N-oxide: a carnitine-derived metabolite that prolongs the hypertensive effect of angiotensin II in rats. Can J Cardiol. 2014;30:1700-5. [9] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335-6. [10] Craciun S, Balskus EP. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:21307-12. [11] Romano KA, Vivas EI, Amador-Noguez D, Rey FE. Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the proatherogenic metabolite trimethylamine-N-oxide. mBio. 2015;6:e02481. [12] Sanders PW. Dietary salt intake, salt sensitivity, and cardiovascular health. Hypertension. 2009;53:442-5. [13] Senthong V, Wang Z, Fan Y, Wu Y, Hazen SL, Tang WH. Trimethylamine N-Oxide and Mortality Risk in Patients With Peripheral Artery Disease. Journal of the American Heart Association. 2016;5. [14] Ufnal M, Zadlo A, Ostaszewski R. TMAO: A small molecule of great expectations. Nutrition. 2015;31:1317-23. [15] Rohrmann S, Linseisen J, Allenspach M, von Eckardstein A, Muller D. Plasma Concentrations of Trimethylamine-N-oxide Are Directly Associated with Dairy Food Consumption and Low-Grade Inflammation in a German Adult Population. The Journal of nutrition. 2016;146:283-9. [16] Obeid R, Awwad HM, Keller M, Geisel J. Trimethylamine-N-oxide and its biological variations in vegetarians. European journal of nutrition. 2016. [17] Cho CE, Taesuwan S, Malysheva OV, Bender E, Tulchinsky NF, Yan J, et al. Trimethylamine-N-oxide (TMAO) response to animal source foods varies among healthy young men and is influenced by their gut microbiota composition: A randomized controlled trial. Molecular nutrition & food research. 2017;61. [18] Cheung W, Keski-Rahkonen P, Assi N, Ferrari P, Freisling H, Rinaldi S, et al. A metabolomic study of biomarkers of meat and fish intake. The American journal of clinical nutrition. 2017;105:600-8. [19] Sleator RD, Hill C. Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS microbiology reviews. 2002;26:49-71. [20] Marshall BJ, Ohye DF, Christian JH. Tolerance of bacteria to high concentrations of NaCl and glycerol in the growth medium. Applied microbiology. 1971;21:363-4. [21] Hung S-C, Yang T-M, Tarng D-C. SP260HIGH SALT DIET ALTERS GUT MICROBIOTA LEADING TO INFLAMMATION AND PROGRESSION OF CKD. Nephrology Dialysis Transplantation. 2017;32:iii194-iii. [22] Sandek A, Bauditz J, Swidsinski A, Buhner S, Weber-Eibel J, von Haehling S, et al. Altered intestinal function in patients with chronic heart failure. Journal of the American College of Cardiology. 2007;50:1561-9. [23] Santisteban MM, Qi Y, Zubcevic J, Kim S, Yang T, Shenoy V, et al. Hypertension-Linked Pathophysiological Alterations in the Gut. Circulation research. 2017;120:312-23.
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[24] Ufnal M, Pham K. The gut-blood barrier permeability - A new marker in cardiovascular and metabolic diseases? Med Hypotheses. 2017;98:35-7. [25] Camilleri M, Madsen K, Spiller R, Greenwood-Van Meerveld B, Verne GN. Intestinal barrier function in health and gastrointestinal disease. Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society. 2012;24:503-12. [26] Treacy EP, Akerman BR, Chow LM, Youil R, Bibeau C, Lin J, et al. Mutations of the flavincontaining monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Human molecular genetics. 1998;7:839-45. [27] Tang WH, Wang Z, Kennedy DJ, Wu Y, Buffa JA, Agatisa-Boyle B, et al. Gut microbiotadependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease. Circulation research. 2015;116:448-55. [28] Yancey PH, Siebenaller JF. Co-evolution of proteins and solutions: protein adaptation versus cytoprotective micromolecules and their roles in marine organisms. The Journal of experimental biology. 2015;218:1880-96. [29] Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. The Journal of experimental biology. 2005;208:2819-30. [30] Somero G. From Dogfish to Dogs: Trimethylamines Protect Proteins from Urea. Physiology. 1986;1:9-12.
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Figure 1. (A) stool concentration, (B) peripheral blood plasma concentration, and (C) 24hr urine excretion in in rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively). Trimethylamine (TMA), trimethylamine N-oxide (TMAO) and indoxyl sulfate (Indoxyl). Values are means, ± SE. ANOVA followed by post-hoc Tuckey-test (* - p < 0.05).
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Figure 2. Alpha diversity analysis: A) Similar number of observed operational taxonomic units (OTUs) in stools of rats maintained on tap water (controls), rats maintained on 0.9% NaCl solution (Isotonic group) and rats maintained on 2% NaCl solution (Hypertonic group) for two weeks. B) No significant differences in species richness among analyzed groups based on Shannon diversity index.
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Figure 3. Principal coordinate analysis of 16S sequences from 26 stool samples using unweighted UniFrac. Rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively).
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Figure 4. The percentage of reads mapped to bacterial genera with TMA producing capacity (Clostridium, Collinsella, Desulfovibrio, Lactobacillus, Proteus). Stool samples form rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively).
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Figure 5. (A) Trimethylamine (TMA) portal blood level (µg/ml) at baseline (0) and 15 min after the intracolonic administration of TMA (1 mg/kg of body weight), (IC TMA). (B) TMA liver clearance at baseline and 15 min after the intracolonic administration of TMA. TMA liver clearance is defined as the percent of TMA metabolized by the liver (1x 100%). Rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively). Values are means, + SE. * - p<0.05 vs baseline (paired T-Test).
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Table 1. Body weight, food intake and water-electrolyte balance in rats maintained either on water (controls) or 0.9 % or 2% NaCl water solution (Isotonic and Hypertonic groups, respectively). Values are means, ± SE. ANOVA followed by post-hoc Tuckey-test. Electrolytes measured only in haemolysis-free plasma samples. † - Controls vs Hypertonic group, ‡ - Isotonic group vs Hypertonic group. Group/
Controls
Isotonic
Hypertonic
ANOVA
358.9±5.0
354.1±3.9
352.1±3.5
ns
409.3±5.9
402.7±5.0
344.4±10.2
(F2,24=23.0,
Parameter Body mass (g), the first day of the experiment Body mass (g), the last
p<0.05) †,‡
day of the experiment Change in body mass (g)
50.3±2.9
48.6±4.8
-7.7±7.8
(F2,24=35.6, p<0.05) †,‡
24hr food intake (g)
28.1±0.8
28.9±0.9
19.4±1.2
(F2,24=25.4, p<0.05) †,‡
24hr water intake (ml)
29.1±1.1
49.8±3.2
139.5±19.7
(F2,24=25.6, p<0.05) †,‡
24hr urine output (ml)
12.2±1.2
22.4±2.4
105.9±17.4
(F2,24=25.5, p<0.05) †,‡
24hr stool output (g)
15.4±0.7
15.7±1.6
9.3±0.8
(F2,24=10.7, p<0.05) †,‡
Stool density (g/ml)
1.14±0.03
1.18±0.04
1.12±0.03
ns
Plasma sodium (g/ml)
3.091±0.016
3.085±0.013
3.364±0.101
(F2,19=7.1, p<0.05)†,‡
Plasma potassium (g/ml)
0.175±0.006
0.174±0.005
0.172±0.012
ns 23 Page 23 of 31
Plasma creatinine (g/ml)
0.0086±0.0006
0.0083±0.0004 0.0076±0.0007 ns
24hr sodium urine
0.043±0.005
0.191±0.016
excretion (g)
1.087±0.227
(F2,24=20.3, p<0.05) †,‡
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Table 2 . Analysis of bacterial genera in stools of rats maintained on tap water (controls), rats maintained on 0.9% NaCl solution (Isotonic group) and rats maintained on 2% NaCl solution (Hypertonic group) for two weeks. The table shows significant differences between controls, Isotonic and Hypertonic groups in bacterial genera, p < 0.05, by Kruskal-Wallis test with FDR multiple test correction. Genus FDR p value Akkermansia
0.006
SMB53
0.006
Proteus
0.006
Facklamia
0.006
Corynebacterium
0.007
Sarcina
0.008
Lachnospira
0.011
Staphylococcus
0.012
02d06
0.016
Aggregatibacter
0.018
Actinomyces
0.018
Helicobacter
0.019
Turicibacter
0.020
Streptococcus
0.023
Acinetobacter
0.027
Rothia
0.030
Aerococcus
0.030
Anaerovibrio
0.034
Candidatus 0.044 Arthromitus Prevotella
0.044
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Jeotgalicoccus
0.044
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Figure 3_color_300dpi.tif
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Figure 4_dopasowana_300dpi.tif
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Figure_1_metabolites.tif
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Figure_2_dopasowana_300dpi.jpg
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Figure_5_bariera_klirens.tif
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