Trimethylamine, a gut bacteria metabolite and air pollutant, increases blood pressure and markers of kidney damage including proteinuria and KIM-1 in rats

The reciprocal interaction between the cardiovascular and renal systems is well established [1]. Furthermore, heart failure often coexists with renal failure, which is defined as a cardiorenal syndrome (CRS). There are several types of CRS, which are categorized by their primary cause (heart or kidney dysfunction) and duration (chronic or acute). Other comorbidities may also be involved in the development of CRS including diabetes mellitus or sepsis [2].

Recently, the association between CRS and gut microbiota has been intensively investigated [3‐6]. For instance, it has been postulated that trimethylamine oxide (TMAO), one of the gut microbiota-derived metabolites, may be involved in the pathogenesis of cardiovascular and kidney diseases. Several studies have shown that patients with chronic kidney disease (CKD) have elevated plasma and/or urine TMAO concentration [7‐10]. Besides, patients with end-stage renal failure show increased plasma TMAO levels, which decreases after hemodialysis [11]. Tang et al. found that TMAO contributes to progressive kidney dysfunction and renal fibrosis [10]. Finally, a positive correlation between high plasma TMAO and atherosclerosis [12‐14], heart failure [15, 16] and hypertension [17, 18] was reported, and a causal relation between TMAO and cardiovascular disease (CVD) has been proposed. However, other studies suggest beneficial effect of TMAO in CVD [19‐21]. Therefore, currently TMAO is considered a biomarker in CVD and renal diseases, but the causative role of TMAO in CVD is open to debate and may depend on TMAO concentration and animal species.

Interestingly, our previous studies found that trimethylamine (TMA), but not TMAO, exerts toxic effects on the cardiovascular system. Specifically, TMA increased blood pressure in anesthetized rats during the intravenous administration and exerted cytotoxic effects in in vitro studies [22‐24].

Plasma TMAO originates from TMA, a gut bacteria product of choline and carnitine [25]. Other origins of TMA in the human body are also possible. First, TMA is manufactured on the order of thousands of tons worldwide. It is used in the production of plastics, disinfectants, insect attractants, intense sweeteners, seafood flavor, vitamin B4 and many other compounds. Second, TMA is an air pollutant [26]. TMA oxidation occurs mainly in the liver by the action of flavin monooxygenase 3 (FMO3) [27]. Both TMA and TMAO are removed from the body by the kidneys [25], although TMA was also detected in exhaled air in patients with end-stage renal disease [28]. The impact of TMA on human health is poorly determined; however, some studies suggested toxic effects of TMA [28‐31].

To the best of our knowledge, the effect of chronic TMA administration on renal and cardiovascular systems has not been evaluated thus far. Therefore, the present study aimed to assess the impact of chronic TMA exposure on cardiovascular and renal systems in rats.

The novel finding of our study is that chronic administration of TMA causes proteinuria, elevated urine KIM-1 and glucose levels, histological characteristics of chronic progressive nephropathy and increased blood pressure in rats. These results suggest a deleterious effect of TMA on the kidneys and cardiovascular system.

Recently, the effects of TMAO on the cardiovascular and renal systems have been researched extensively [36, 37]. Some studies suggest that TMAO may contribute to the development of cardiorenal syndrome [3, 4]. On the other hand, it has been well established that TMAO is one of the osmolytes protecting proteins from high osmotic pressure [38, 39]. For instance, TMAO and other osmolytes such as betaine, sorbitol and glycerophosphorylcholine play a protective role in the renal medulla in which osmolality exceeds plasma osmolality by up to 4–5-folds [40‐42].

In our study, chronic administration of TMA caused proteinuria, glucosuria and elevated KIM-1 levels in urine (Fig. 2). Proteinuria is a well-known marker of kidney damage [43, 44], and is associated with CVD [45, 46]. KIM-1 is a transmembrane tubular protein, which is up-regulated and present in urine after the renal tubular injury, both in rats and humans [47, 48]. This protein is also a marker for drug-induced kidney toxicity [49]. Glucosuria is a common finding in diabetes mellitus and diabetic kidney disease but it may also be present in nondiabetic advanced CKD [50]. In contrast to humans, in rats glucose is observed also in healthy animals [51‐53]. Notably, in this study the urine glucose concentration was significantly higher in the TMA groups than in the control group, which further points to tubular damage in rats exposed to TMA in drinking water. The chronic administration of TMA also caused histopathological changes, including thickening of the basement membranes and increased infiltration of mononuclear cells without signs of concomitant inflammation, which may reflect CPN (Fig. 4). CPN is a spontaneous disease with unknown etiology. The observed changes in the renal parenchyma in rats receiving TMA may result from either the activation or aggravation of the ongoing CPN due to a direct effect of TMA on the kidneys or may be secondary to TMA-induced hemodynamic disturbances [54‐56].

The association between CKD and hypertension is widely studied and hypertension may be a cause and a consequence of kidney disease [57‐59]. Herein we observed that rats administered TMA showed increased arterial blood pressure (Table 2). Specifically, a significant increase in systolic blood pressure was accompanied by a more moderate increase in diastolic blood pressure. Systemic vascular resistance calculated from cardiac output and mean arterial blood pressure tended to be higher in rats exposed to the higher dose of TMA, which suggest that in this group the increase in BP was in part mediated by vasoconstriction. In general, these findings align with our previous studies, showing hypertensive response after the acute administration of TMA [22]. In this study, we did not find indications of fluid retention or water-electrolyte disturbances. However, rats on TMA tended to drink more water and show elevated serum potassium levels. Renal regulation of water-electrolyte balance and blood pressure is exerted mainly through the renin-angiotensin system. However, we did not find any consistent changes in the RAS gene expression in the kidneys that could explain the effect of TMA on blood pressure and water-electrolyte balance (Additional file 5: Fig. S1). Finally, we did not find any significant changes in echocardiographic parameters (Additional file 3: Table S2). Specifically, TMA and control groups did not differ in the atria and ventricle size, stroke volume or ejection fraction. Therefore, it seems that the magnitude or/and duration of hemodynamic changes were insufficient to produce noticeable remodeling of the heart.

There are some clinical data linking high TMA levels to renal system pathologies. Hsu et al. showed that children with end-stage renal failure had higher plasma concentration of TMA than patients with stage 1 chronic kidney disease. They also demonstrated that plasma TMA levels were inversely correlated with high BP and eGFR [8]. Research also shows that patients with end-stage renal disease have increased plasma TMA concentration [11] and TMA levels in exhaled air [28], but these levels decrease after dialysis. There is also evidence that patients with trimethylaminuria due to FMO3 deficiency show higher blood pressure [60].

However, the effects of TMA on the renal and cardiovascular systems have not been studied so far in interventional studies. To the best of our knowledge, our study is the first showing the negative effect of TMA on the kidneys; a finding which suggests a causative relationship between TMA accumulation due to kidney failure and cardiorenal complications of CKD. It may be speculated that TMA is one of the mediators in the vicious cycle of the cardiorenal syndrome.

Although there are multiple studies evaluating TMAO and TMA concentrations in fish and other marine animals’ muscle and liver tissues [39, 61‐64], studies showing TMAO and TMA concentration in tissue of mammals are scant or lacking. Present work broadens the knowledge of TMA and TMAO tissue distribution in mammals. In the current study, control animals showed the highest TMA and TMAO concentrations in the renal cortex (158.5 µM/kg and 14.5 µM/kg, respectively) and renal medulla (193.1 µM/kg and 18.5 µM/kg, respectively, Table 4). The lowest TMA and TMAO concentrations were found in the heart (0.7 µM/kg and 3.22 µM/kg, respectively). The liver contained higher levels of TMA (42.8 µM/kg) than TMAO (5.4 µM/kg). TMA concentration (6.5 µM/kg) in the lungs was lower than TMAO concentration (9.3 µM/kg).

Three decades ago, Da Costa et al. assessed TMA and TMAO concentrations in the rat’s liver and kidney [65] but using different methods. They measured concentrations in tissue wet mass, not in tissue dry mass as in our study, making the comparison of Da Costas and our results difficult. TMA concentration was higher in the liver (437 nM/g) and kidney in the Da Costa study (531 nM/g) than in our study. TMAO concentration in the liver was over 100-fold higher (633 nM/g) than in our study, while TMAO concentration in the kidneys was not provided by Da Costa et al. It needs to be stressed that, in contrast to relatively simple and reproducible measurements of TMAO levels across numerous studies [4, 8, 10, 14, 16, 18‐21, 66], the measurements of TMA are much more difficult. There are discrepancies in reported absolute TMA levels across the few available studies [11, 23, 24, 33, 35, 65, 67‐69].

It needs to be noted, that the determination of small volatile amines such as TMA or ammonium is very challenging [67]. In this study, we used the method based on the derivatization technique which gives different TMA plasma results than the methods used before [21, 33]. We suspect that the method without derivatization gives higher plasma levels of TMA due to the presence of analytes containing TMA in their structure which coelute with trimethylamine and break down at the ion source yielding falsely TMA results. After several years of experience with TMA and TMAO determination in various tissues, we believe that the derivatization method is more appropriate because it allows reducing the volatility of trimethylamine and transforms trimethylamine into more stable and more compatible to liquid chromatography derivative before injection. For a detailed description of the method, see Additional file 1: Methods.

After crossing the gut-blood barrier, TMA is metabolized in the liver to TMAO by FMOs, mainly FMO3 [27, 70]. Five isoforms of FMOs are expressed in humans and animals. FMO3 shows the highest expression in the human liver whereas the human kidneys show the highest expression of FMO1 [27]. Rats also show FMO1 and FMO3 expression in the liver and kidneys [71‐73]. Novick et al. observed higher FMO1 and FMO3 expression in the kidneys than in the liver. FMO1 expression was highest in the proximal and distal tubules, whereas FMO3 expression was highest in the distal tubules, collecting tubules and glomeruli [73].

To the best of our knowledge, our study is the first to evaluate the expression of FMO1, FMO3 and FMO5 after chronic TMA administration (Fig. 3). Our study shows that chronic TMA administration reduces FMO3 expression in the renal cortex and FMO1 and FMO3 expression in the renal medulla. Due to the accumulation of products, the negative feedback mechanism is often found in the regulation of enzyme, hormone and receptor activity in human and animal organisms [74‐78]. Therefore, we speculate that rapid oxidation of TMA to TMAO caused downregulation of FMO1 and FMO3 expression in the kidney. Future research on TMA to TMAO oxidation in the kidneys is needed.

Finally, our study, suggests that TMA is rapidly oxidized to TMAO, which may limit the toxic effect of TMA on other organs. Namely, serum TMA concentrations in all groups were at least 100 times lower than TMAO concentrations. Furthermore, administration of TMA resulted in small increases in TMA tissue levels but 7–30-fold increases in TMAO levels (depending on the type of tissue, for comparison of the control group and the H group). Moreover, 24-h urine excretion of TMAO was 11 and 24 times higher in TMA-treated rats (the L group and the H group, respectively). In contrast, TMA 24-h urine excretion increased 17 times only for the H group (in the L group, we did not notice any changes).

In conclusion, chronic TMA administration at high doses increases blood pressure and causes deleterious effects on the kidneys, including proteinuria, increased urine KIM-1 and glucose levels in rats. TMA is rapidly oxidized to TMAO, which may limit the toxic effect of the molecule. Furthermore, TMA lowers the expression of FMO1 and FMO3 in the kidneys. More research on TMA, in particular in pathological states characterized by impaired TMA to TMAO oxidation, is needed to understand the role of TMA and TMAO in the pathological process of cardiorenal syndrome.