Spontaneously hypertensive rats exhibit increased liver flavin monooxygenase expression and elevated plasma TMAO levels compared to normotensive and Ang II-dependent hypertensive rats

Background: Flavin monooxygenases (FMOs) are enzymes responsible for the oxidation of a broad spectrum of exogenous and endogenous amines. There is increasing evidence that trimethylamine (TMA), a compound produced by gut bacteria and also recognized as an industrial pollutant, contributes to cardiovascular diseases. FMOs convert TMA into trimethylamine oxide (TMAO), which is an emerging marker of cardiovascular risk. This study hypothesized that blood pressure phenotypes in rats might be associated with variations in the expression of FMOs. Methods: The expression of FMO1, FMO3, and FMO5 was evaluated in the kidneys, liver, lungs, small intestine, and large intestine of normotensive male Wistar-Kyoto rats (WKY) and two distinct hypertensive rat models: spontaneously hypertensive rats (SHRs) and WKY rats with angiotensin II-induced hypertension (WKY-ANG). Plasma concentrations of TMA and TMAO were measured at baseline and after intravenous administration of TMA using liquid chromatography-mass spectrometry (LC-MS). Results: We found that the expression of FMOs in WKY, SHR, and WKY-ANG rats was in the descending order of FMO3 > FMO1 >> FMO5. The highest expression of FMOs was observed in the liver. Notably, SHRs exhibited a significantly elevated expression of FMO3 in the liver compared to WKY and WKY-ANG rats. Additionally, the plasma TMAO/TMA ratio was significantly higher in SHRs than in WKY rats. Conclusion: SHRs demonstrate enhanced expression of FMO3 and a higher plasma TMAO/TMA ratio. The variability in the expression of FMOs and the metabolism of amines might contribute to the hypertensive phenotype observed in SHRs.

Interestingly, high TMAO concentrations has been suggested to corelate with increased cardiovascular risk (Tang et al., 2013;Qi et al., 2018).The blood TMAO level has been reported to be positively correlated with long-term mortality risk in patients with atherosclerosis, heart failure, and chronic kidney disease (Koeth et al., 2013;Tang et al., 2014;Tang et al., 2015).
We hypothesized that the hypertensive rat phenotype might be linked to changes in the expression and activity of FMOs.Consequently, the main aim of our study was to compare the expression of FMOs in normotensive and hypertensive rats.We carried out this experiment using two different models of hypertension: the genetic SHR model and the pharmacologically induced model using Ang II.Rats were housed in groups of two to three animals, in polypropylene cages with environmental enrichment, 12 h light/ 12 h dark cycle, temperature 22-23 °C, humidity 45%-55%, food and water ad libitum.12-week-old, male.

Blood pressure measurement
Before the experiment, blood pressure was recorded in rats anaesthetized with urethane (1.5 g/kg intraperitoneally, Sigma-Aldrich, Poland) via a polyurethane catheter inserted into the femoral artery.Haemodynamics were recorded using Biopac MP 160 system (Biopac Systems, Goleta, CA, United States).Blood pressure was assessed as a baseline prior to the intravenous infusion of TMA.

Gene and protein expression
12-week-old WKY, SHR and WKY-Ang II rats were killed, tissues samples were collected and frozen immediately.Real-time PCR was used to detect FMO1, FMO3 and FMO5 gene expression in the kidney medulla, kidney cortex, liver, lungs, small intestine and colon.

Real-time PCR
In short, about 20 mg of every tissue was homogenized on BeadBug ™ microtube homogenizer (Benchmark Scientific, Inc.).
Total RNA was isolated from samples according to TRI Reagent ® protocol.cDNA was transcribed from RNA samples according to iScript ™ Reverse Transcription Supermix protocol (Bio-Rad).The qPCR mixes were prepared according to the Bio-Rad SsoAdvanced ™ universal SYBR ® Green Supermix protocol.Amplifications were performed in a Bio-Rad CFX Connect Real-Time System under standardized conditions using commercial assays.

Western blot
For the analysis of target proteins, total protein extracts were prepared from the, liver,.In short, frozen samples were suspended in a histidine-sucrose buffer (30 mM histidine, 250 mM sucrose, 2 mM EDTA, proteases inhibitors, PMSF, pH 7.4), homogenized, centrifuged (10,000 RCF, 10 min, 4 °C).After removing the supernatant, 150 µL of lysis buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 2% Triton-X, proteases inhibitors) was added to the pellet and resuspended by vortexing.The supernatant was separated for protein concentration analysis using a Bradford Protein Assay (Bio-Rad, Hercules, CA, United States).For all Western blot analyses, a 4× Laemmli sample buffer was added to samples.To determinate the levels of FMO1, FMO3 and FMO5 all samples were resolved by electrophoresis on 12% SDS/PAGE gels.Resolved proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA, United States), blocked using skim milk and incubated with primary and secondary antibodies.For quantitative analysis of protein content, reactive bands were quantified relative to those of actin using a ChemiDoc MP Imaging System, Densitometric analysis was performed using Quantity One software version 4.6.8(Bio-Rad, Hercules, CA, United States).Uncropped blots and list of antibodies are presented in Supplementary Figure S5 and Supplementary Table S1.
Blood samples from femoral vein, were collected at baseline, 10 min and 20 min after the intravenous infusion of TMA at a dose of 45 μmol/kg, 135 μmol/kg or 405 μmol/kg.
Plasma concentrations of TMA and TMAO was evaluated using Waters Acquity Ultra Performance Liquid Chromatograph coupled with Waters TQ-S triple-quadrupole mass spectrometer.Samples were prepared using the derivatization technique based on Johnson's protocol with modification (Johnson, 2008).The mass spectrometer was operated in multiple-reaction monitoring (MRM)-positive electrospray ionization (ESI+) mode for all analytes.The concentrations of analytes were calculated using calibration standard mix derived from a series of calibrator samples by spiking standard stock solutions into water.Plasma samples were compared with an obtained calibration curve.

Statistics
The Kolmogorov-Smirnov test was used to test normality of the distribution.
To evaluate changes in pharmacokinetic data in response to treatment, baseline values were compared with post-treatment values using one-way analysis of variance (ANOVA) for repeated measures.This was followed by Tukey's post hoc test for multiple comparisons to identify differences between baseline and post-dose time points.Differences between groups/series were assessed using multivariate ANOVA, followed by Tukey's post hoc test or by a t-test, as appropriate.A two-sided p-value of less than 0.05 was considered statistically significant.Analyses were performed using GraphPad Prism version 8.4.3 (GraphPad Software Inc., San Diego, CA, USA).Sample size calculation for Fmo's analysis was conducted using G*Power software version 3.1.9.7, estimating a minimum required number of animals per group to be 6.Measurements was determined based on the following assumed parameters: difference between subjects (groups) 40% population mean 10 arbitrary unit (a.u) common standard deviation 0.9, for alpha error 0.05, test power 0.8.The post hoc power analysis was performed for significant differences by utilizing the online calculator: https:// clincalc.com/stats/Power.aspx(Supplementary Table S2).The analysis of false discovery rate (FDR) for FMO3 mRNA and protein expression was conducted (Supplementary Tables S3, S4).
In general, all the groups, independently on tissue type, showed the gene expression of FMOs subfamilies in the following order of magnitude FMO3>FMO1>>FMO5 (Figure 1).With regard to tissue distribution of FMOs gene expression, high expression of FMOs was found in the liver, lungs and kidneys, whereas low FMOs expression was present in small intestine and colon.In relation to the liver's most abundant mRNA FMO's expression, we have conducted comprehensive investigations aimed at identifying the FMOs in this organ at the protein level.

Hepatic mRNA and protein expression of FMOs
In the liver, there was notably elevated mRNA expression of FMO3 in SHR compared to WKY (p < 0.01), while FMO1 and FMO5 exhibited no significant differences between the two strains.Interestingly, the WKY-ANG group showed significantly higher expression levels of FMO3(p < 0.01) and FMO5 (p < 0.05) than WKY strain (Figure 2 A).
All statistical comparisons were made against WKY which was a control group in all gene and protein-based experiments.
Infusion of TMA produced a significant, dose-dependent increase in plasma TMA and TMAO in all the groups.The increase in plasma TMAO was more rapid in SHR than in the other groups (Supplementary Figures S1-S4).
SHR group showed significantly higher plasma TMAO/TMA ratio than WKY and WKY-ANG 10 min after the infusion of TMA at a dose of 45 μmol/kg, whereas 20 min after the infusion, SHR showed significantly higher plasma TMAO/TMA ratio than WKY and WKY-ANG, for all TMA doses, i.e. 45, 135 and 405 μmol/kg (Figures 4A-C).

Discussion
The novel finding of our study is that SHRs show higher hepatic gene expression and protein levels of FMOs and more rapid oxidation of TMA to TMAO.
In the present study we evaluated two animal models of hypertension: SHRs and WKY-ANG.The SHR strain, derived from WKY rats, is the most commonly used animal model for essential hypertension in humans (Louis and Howes, 1990).
SHRs begin to develop hypertension between the fourth and sixth weeks of age, and by the 10th week of life, their arterial blood pressure is 30% higher than that of WKY rats (Kokubo et al., 2005;Koga et al., 2008).Blood pressure measurements in anesthetized rats in this study revealed higher mean arterial blood pressure in both SHR and WKY-ANG rats, confirming their hypertensive phenotype.
Here, we found that WKY, SHR and WKY-ANG show expression of the three subfamilies of FMO in the following order of magnitude FMO3>FMO1>>FMO5.Furthermore, we found that FMOs are expressed in the following tissues: liver, kidney, lungs, colon and intestines, with the greatest expression of FMOs was found in the liver.
In general, the most significant differences in gene and protein expression of FMOs and the pharmacokinetics of TMA were observed between the WKY and SHR, with WKY-ANG rats displaying characteristics that were a blend of both WKY and SHR strains.Specifically, compared to WKY, SHR exhibited significantly higher liver protein expression across all subfamilies of FMOs, whereas WKY-ANG rats showed an increase only in FMO5.Importantly, the elevated expression of FMOs in SHR was linked to a more efficient and rapid oxidation of TMA to TMAO following the intravenous infusion of the amine.This was evidenced by SHRs demonstrating a significantly higher TMAO/TMA ratio after the administration of TMA in increasing doses.Lastly, SHRs also exhibited significantly higher baseline levels of TMAO, corroborating the findings of previous research (Huc et al., 2018).This study, suggests that greater oxidation of TMA to TMAO in SHRs may contribute to higher plasma TMAO levels in hypertensive rats, in addition to previously described factors such as increased gut-bloodbarrier permeability to bacterial metabolites including TMA in hypertensive intestines (Jaworska et al., 2017;Drapala et al., 2020).Some research suggest that alterations in FMOs expression are associated with several diseases including trimethylaminuria (TMAU) (Montoya Alvarez et al., 2009), diabetes mellitus (Rouer et al., 1988;Siddens et al., 2014), familial adenomatous polyposis (Cruz-Correa and Giardiello, 2003), breast (Krueger et al., 2006), prostate (Mondul et al., 2015) and colorectal cancer (Xie et al., 2012), peptic ulcer and gastro-oesophageal reflux (Chung et al., 2000) and hemochromatosis (Muckenthaler et al., 2003).Furthermore, some evidence suggests that patient with trimethylaminuria show higher blood pressure and exaggerated response to pressor amines like tyramine and phenethylamine (Forrest et al., 2001;Cashman et al., 2003), however, data are not consistent (Dolan et al., 2005;D'Angelo et al., 2013).There is also limited data on FMO3 polymorphisms and its effect on hypertension, but studies provide conflicting results (Akerman et al., 1999;Cashman et al., 2000;Cashman et al., 2003;Dolan et al., 2005;D'Angelo et al., 2013).Finally, some links between blood pressure and inactivation of biogenic amines by FMO3 (Cashman et al., 1997;Lin and Cashman, 1997;Treacy et al., 1998;Cashman et al., 2000) exist.
In the scientific literature, various models of hypertension are well-documented.For our research, we chose two models that are widely recognized and extensively used to represent human hypertension.This selection was influenced by the unique and differing etiologies of hypertension presented by these models, as well as their widespread acceptance as representative models for studying human hypertension (Jama et al., 2022).The presence of numerous underlying mechanisms driving hypertension underscores the critical need for future research to use alternative models for more comprehensive exploration.
The limitation of this study arises from its exclusive use of male rats, a decision aimed at minimizing biological variability due to hormonal fluctuations, which are known to significantly impact small experimental study outcomes.For future research, it is crucial to consider the inclusion of both sexes to ensure a more comprehensive understanding of TMA metabolism and FMOs activity in hypertensive rats.Additionally, measuring FMO expression in the heart, brain, and blood vessels would be beneficial, considering their potential impact on blood pressure and blood flow regulation within these tissues.
In conclusion, this study offers a comprehensive demonstration of the relationship between hepatic FMO expression and the oxidation of TMA to TMAO in the two animal models of hypertension.Our results indicate that hypertension in SHRs is linked to an increased expression and activity of liver FMOs.Further experimental research is necessary to clarify the role of FMOs in the pathogenesis of cardiovascular diseases.The findings from this study lay the groundwork for subsequent investigations into FMOs as a potential therapeutic target for hypertension treatment.

Data availability statement
The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.Plasma TMAO/TMA ratio in WKY, SHR and WKY-ANG rats after intravenous administration of TMA at a dose of 45 (A), 135 (B) and 405 (C) μmol/kg.Two way ANOVA *p < 0.05 SHR vs WKY, † p < 0.05 SHR vs WKY-ANG.

FIGURE 2 (
FIGURE 2 (A) RT-qPCR analysis of FMO1 FMO3 and FMO5 transcript levels in the liver of WKY, SHR and WKY-ANG rats (displays on histogram use arbitrary units).(B) FMO1, FMO3 and FMO5 protein levels in the liver examined by Western blot analysis.Beta-actin and the Ponceau-S staining were used as a reference for equal protein loading control.Quantification of the band intensity of protein expression was performed using Quantity One software The relative levels of the test proteins are plotted in arbitrary unit (means ± SD). (C) Representative blots of hepatic FMO's protein of WKY, SHR and WKY-ANG rats.*p < 0.05 vs. WKY, **p < 0.01 vs. WKY #p < 0.05 SHR vs. WKY-ANG.