Metabolic parameters:
After the treatment, there were no significant differences between the groups in body weight, food intake and urine output. Rats on TMA tended to drink more water and excrete more feces (Table 1).
Table 1. Metabolic parameters
Parameter
|
Control group
|
L group
|
H group
|
One-way ANOVA
|
Body weight [g]
|
431.52 (±20.08)
|
429.77 (±25.56)
|
440.93 (±21.22)
|
P = 0.53
|
Water [g/24hrs]
|
31.94 (±6.01)
|
36.28 (±5.07)
|
35.47 (±5.04)
|
P = 0.21
|
Food [g/24hrs]
|
19.28 (±1.85)
|
20.23 (±1.79)
|
20.33 (±2.70)
|
P = 0.53
|
Faces [g/24hrs]
|
7.74 (±2.33)
|
10.08 (±2.38)
|
9.48 (±1.66)
|
P = 0.08
|
Urine [g/24hrs]
|
19.17 (±5.64)
|
19.32 (±3.53)
|
18.83 (±3.75)
|
P = 0.97
|
Abbreviations: L group – TMA low-dose group; H group – TMA high-dose group. All data are expressed as the mean ± SD. ANOVA followed by post hoc Tuckey-test.
Hemodynamic and ECHO parameters
Rats on TMA showed higher blood pressure (Table 2). Specifically, the H group had significantly higher systolic blood pressure and demonstrated a trend towards higher diastolic blood pressure. TMA rats also tended to have higher HR.
There were no significant differences between the groups in echocardiographic parameters (Table 3).
Table 2. Hemodynamic parameters
Parameter
|
Control group
|
L group
|
H group
|
One-way ANOVA
|
SBP [mmHg]
|
126.3 (±11.4)
|
141.3 (±17.9)
|
151.2 (±19.9)*
|
P = 0.02
|
DBP [mmHg]
|
70.8 (±14.0)
|
64.2 (±13.5)
|
81.78 (±14.1)
|
P = 0.06
|
Heart rate [bpm]
|
314 (±86)
|
330 (±58)
|
373 (±13)
|
P = 0.16
|
SVR [mmHg⋅min⋅mL-1]
|
1.51 (±0.95)
|
0.73 (±0.19)
|
3.67 (±5.16)
|
P = 0.14
|
Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; SVR, Systemic Vascular Resistance; L group – TMA low-dose group; H group – TMA high-dose group. All data are expressed as the mean ±SD; ANOVA followed by post hoc Tuckey-test; *P < 0.05 vs. control group.
Table 3 – Echocardiographic parameters
Parameter
|
Control group
|
L group
|
H group
|
One-way ANOVA
|
IVSD [cm]
|
0.21 (±0.03)
|
0.20 (±0.04)
|
0.24 (±0.05)
|
P = 0.18
|
LVDD [cm]
|
0.53 (±0.11)
|
0.62 (±0.11)
|
0.52 (±0.16)
|
P = 0.26
|
PWD [cm]
|
0.24 (±0.03)
|
0.24 (±0.03)
|
0.27 (±0.03)
|
P = 0.05
|
IVSS [cm]
|
0.29 (±0.04)
|
0.26 (±0.05)
|
0.32 (±0.06)
|
P = 0.09
|
LVDS [cm]
|
0.34 (±0.08)
|
0.39 (±0.11)
|
0.34 (±0.11)
|
P = 0.49
|
PWS [cm]
|
0.32 (±0.03)
|
0.32 (±0.04)
|
0.35 (±0.03)
|
P = 0.06
|
EF [%]
|
71.11 (±7.37)
|
71.44 (±12.61)
|
68.57 (±9.98)
|
P = 0.81
|
FS [%]
|
35.33 (±5.61)
|
38.22 (±9.08)
|
34.16 (±8.67)
|
P = 0.54
|
LVEDV [ml]
|
0.39 (±0.22)
|
0.54 (±0.27)
|
0.40 (±0.26)
|
P = 0.40
|
LVESV [ml]
|
0.11 (±0.07)
|
0.18 (±0.13)
|
0.13 (±0.08)
|
P = 0.27
|
SV [ml]
|
0.28 (±0.16)
|
0.42 (±0.18)
|
0.28 (±0.20)
|
P = 0.29
|
AO [cm]
|
0.40 (±0.05)
|
0.42 (±0.04)
|
0.38 (±0.06)
|
P = 0.39
|
LA [cm]
|
0.42 (±0.05)
|
0.45 (±0.06)
|
0.45 (±0.05)
|
P = 0.48
|
CO [ml/min]
|
87.54 (±64.27)
|
132.45 (±45.57)
|
92.49 (±69.45)
|
P = 0.28
|
Abbreviations: IVSD, interventricular septum thickness at diastole; LVDD, left ventricular diastolic diameter; PWD, posterior wall thickness at diastole; IVSS, interventricular septum thickness at systole; LVDS, left ventricular diastolic diameter; PWS, posterior wall thickness at systole; EF, ejection fraction; FS, fractional shortening, LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; AO, aortic root diameter; LA, left atrium diameter; CO; cardiac output; L group – TMA low-dose group; H group – TMA high-dose group. All data are expressed as the mean ± SD; ANOVA followed by post hoc Tuckey-test.
TMA and TMAO concentrations in stool, serum and urine
The median TMA stool concentration was significantly higher in the H group than in the control or L group. The median TMA and TMAO serum concentrations were significantly higher in the H group than in the control group, 8-fold and 6-fold, respectively. TMA and TMAO serum concentrations were also higher in the L group than in the control group. The median TMA and TMAO urine concentrations were significantly higher in the H group than in controls, 17-fold and 24-fold, respectively. The median TMAO urine concentration was higher in the L group than in the control group (11-fold), but TMA urine concentration was similar to the control group (Table 4).
There were no significant differences between groups in choline and carnitine concentrations. TMAO concentration in stool was below the lower limit of quantification.
Table 4. TMAO, TMA and their precursors concentrations in stool, serum and urine.
Parameter [µM/l]
|
Control group
|
L group
|
H group
|
Kruskal-Wallis test
|
Stool
TMA
|
19.70 (13.86; 30.38)
|
30.14 (21.27; 41.67)
|
54.62 (47.88; 79.21)**
|
P = 0.003
|
Choline
|
77.55 (67.81; 92.10)
|
61.87 (57.19; 84.98)
|
81.52 (77.15; 88.23)
|
P = 0.41
|
Carnitine
|
8.91 (7.43; 11.49)
|
6.07 (3.97; 8.03)
|
6.52 (5.87; 7.62)
|
P = 0.26
|
Serum
TMAO
|
8.96 (6.46; 11.85)
|
26.25 (25.30; 31.67)*
|
49.50 (45.84; 84.76)***
|
P <0.001
|
TMA
|
0.04 (0.03; 0.04)
|
0.14 (0.09; 0.17)*
|
0.32 (0.27; 0.64)***
|
P < 0.001
|
Choline
|
75.92 (60.91; 104.79)
|
117.31 (93.88; 135.97)
|
95.77 (78.62; 110.24)
|
P = 0.13
|
Carnitine
|
66.77 (60.34; 70.64)
|
74.90 (64.88; 80.64)
|
66.47 (57.60; 73.78)
|
P = 0.29
|
Urine
TMAO
|
584.98 (448.50; 686.60)
|
6 143.59 (5 569.76; 6 449.26)*
|
14 049.76 (13 902.77; 15 586.82)***; #
|
P < 0.001
|
TMA
|
15.18 (9.75; 20.99)
|
15.10 (12.94; 16.37)
|
257.37 (207.48; 324.83)***; ###
|
P <0.001
|
Choline
|
94.41 (69.78; 151.54)
|
135.07 (118.75; 160.19)
|
137.25 (129.10; 154.52)
|
P = 0.27
|
Carnitine
|
169.45 (142.06; 232.12)
|
153.41 (141.67; 171.76)
|
134.18 (123.83; 165.68)
|
P = 0.23
|
Abbreviations: TMA, trimethylamine; TMAO, trimethylamine oxide; L group – TMA low-dose group; H group – TMA high-dose group. All data are expressed as the median (Q1; Q3); Kruskal-Wallis test followed by post-hoc Dunn’s test. *P < 0.05 vs. control group; ***P < 0.001 vs. control group; #P < 0.05 vs. low dose group; ###P < 0.001 vs. low dose group.
TMA/TMAO balance
L and H groups consumed TMA in water at an average dose of 200 and 500 µM/day, respectively. The amount of TMA excreted per day was significantly higher in the H group than in the control or L group, 17-fold and 20-fold, respectively. The amount of TMAO excreted per day was significantly higher in H and L groups than in the control group (Fig. 1), 24-fold and 11-fold, respectively.
Figure 1 title: TMA/TMAO balance
Figure 1. TMA/TMAO balance. (a) the amount of TMA consumed per day; (b) the amount of TMA excreted per day; (c) the amount of TMAO excreted per day in rats maintained either on tap water (control group) or low and high dose of TMA. Abbreviations: TMA, trimethylamine; TMAO, trimethylamine oxide. All data are expressed as the median (n = 9); Kruskal-Wallis test followed by post-hoc Dunn’s test; ***P < 0.001 vs. tap water group; ###P < 0.001 vs. low dose group.
TMA and TMAO concentrations in the tissue
In most of the evaluated tissues, TMAO concentrations were several-fold higher in TMA groups than in controls. In contrast, TMA levels were only moderately higher in TMA groups and there were no significant differences in choline and carnitine levels. (Table 5).
Table 5. The TMAO, TMA and other metabolites concentrations in the tissue
Parameter [µM/kg]
|
Control group
|
L group
|
H group
|
Kruskal-Wallis test
|
Lungs
TMAO
|
9.27 (5.19; 11.07)
|
52.04 (48.16; 57.69)*
|
218.25 (191.74; 282.54)***
|
P < 0.001
|
TMA
|
6.52 (5.14; 8.93)
|
6.71 (5.85; 8.63)
|
9.76 (7.26; 11.28)
|
P = 0.18
|
Choline
|
1 870.59 (1 784.86; 2 062.80)
|
2 038.94 (1 985.86; 2 159.95)
|
1 969.03 (1 877.74; 2 120.19)
|
P = 0.42
|
Carnitine
|
307.11 (277.93; 309.62)
|
279.62 (272.50; 342.72)
|
309.68 (293.66; 334.90)
|
P = 0.23
|
Liver
TMAO
|
5.40 (4.24; 9.57)
|
19.82 (13.79; 23.19)
|
35.85 (29.32; 41.87)***
|
P = 0.001
|
TMA
|
42.80 (38.09; 47.25)
|
63.12 (53.61; 73.26)*
|
80.39 (62.98; 92.61)***
|
P < 0.001
|
Choline
|
1 112.92 (921.26; 1 462.16)
|
1 124.02 (814.50; 1 241.82)
|
1 265.63 (1 014.44; 2 007.82)
|
P = 0.11
|
Carnitine
|
470.73 (452.88; 481.55)
|
514.03 (477.57; 589.24)
|
568.35 (498.70; 589.18)
|
P = 0.34
|
Renal cortex
TMAO
|
14.48 (13.13; 21.87)
|
27.76 (26.04; 36.82)
|
189.74 (82.22; 204.48)***
|
P < 0.001
|
TMA
|
158.48 (149.59; 163.61)
|
240.14 (229.81; 262.72)*
|
487.66 (389.55; 593.86)***
|
P < 0.001
|
Choline
|
1 441.73 (1 159.19; 1 538.25)
|
1 512.66 (1 462.91; 1 687.80)
|
1 346.40 (1 025.19; 1 521.07)
|
P = 0.56
|
Carnitine
|
314.85 (291.49; 362.62)
|
344.41 (226.85; 364.37)
|
358.71 (306.06; 434.33)
|
P = 0.78
|
Renal medulla
TMAO
|
18.53 (14.43; 27.82)
|
42.74 (34.16; 43.01)
|
192.02 (73.95; 213.70)***, #
|
P < 0.001
|
TMA
|
193.08 (186.32; 218.50)
|
286.22 (267.48; 294.91)
|
449.83 (348.32; 524.00)***
|
P < 0.001
|
Choline
|
2 408.67 (1 737.76; 2 570.56)
|
1 198.82 (399.11; 1 493.18)
|
1 751.37 (1 308.89; 2 353.36)
|
P = 0.06
|
Carnitine
|
341.55 (314.27; 355.18)
|
297.82 (288.73; 310.64)
|
306.90 (299.75; 355.56)
|
P = 0.10
|
Heart
TMAO
|
3.22 (2.45; 5.77)
|
35.59 (34.37; 48.97)*
|
96.30 (90.30; 112.43)***
|
P < 0.001
|
TMA
|
0.70 (0.58; 0.85)
|
4.02 (3.67; 4.36)*
|
11.32 (10.42; 13.17)***, #
|
P < 0.001
|
Choline
|
129.98 (124.15; 156.68)
|
145.91 (134.83; 150.98)
|
140.36 (135.37; 152.92)
|
P = 0.45
|
Carnitine
|
759.22 (727.41; 796.04)
|
795.12 (698.43; 838.37)
|
883.12 (814.61; 916.01)
|
P = 0.06
|
Abbreviations: TMA, trimethylamine; TMAO, trimethylamine oxide; L group – TMA low-dose group; H group – TMA high-dose group. All data are expressed as the median (Q1; Q3); Kruskal-Wallis test followed by post-hoc Dunn’s test. *P < 0.05 vs. control group; ***P < 0.001 vs. control group; #P < 0.05 vs. low dose group.
Urine biochemical analysis
Rats from the H group showed significantly higher protein concentration in urine and protein/ creatinine ratio than the control or L group (Fig. 2). Urine KIM-1 levels were significantly higher in the H group compared to the control group.
Figure 2 title. Urine biochemical analysis
Figure 2. Urine biochemical analysis. (a) protein concertation in urine; (b) protein to creatinine ratio in urine; (c) Kim-1 concertation in urine in rats maintained either on tap water (control group) or low and high dose of TMA. Abbreviation: TMA, trimethylamine; UPCR, urine protein creatinine ratio. All data are expressed as the median (n = 9); Kruskal-Wallis test followed by post-hoc Dunn’s test for urine protein and UPCR; ANOVA followed by post-hoc Tuckey test for urine Kim-1; *P < 0.05 vs. tap water group.
Serum biochemical analysis
There was a trend for a higher serum potassium concentration in the TMA groups than in the control group (Table 6). There were no significant differences between groups in the serum urea, serum creatinine, serum sodium concentrations, creatinine clearance and KIM-1 protein in serum.
Table 6. Serum biochemical analysis
Parameter
|
Control group
|
L group
|
H group
|
One-way ANOVA
|
Urea [mg/dl]
|
91.56 (±12.55)
|
79.86 (±17.09)
|
87.00 (±15.87)
|
P = 0.33
|
Creatinine [mg/dl]
|
0.86 (±0.27)
|
0.93 (±0.31)
|
0.88 (±0.19)
|
P = 0.84
|
Sodium [mg/dl]
|
312.33 (±14.41)
|
318.43 (±16.47)
|
325.44 (±23.74)
|
P = 0.35
|
Potassium [mg/dl]
|
18.80 (±1.69)
|
21.66 (±2.58)
|
20.70 (±2.62)
|
P = 0.06
|
Creatinine clearance [ml/min]
|
0.92 (±0.73)
|
1.30 (±0.77)
|
0.99 (±0.42)
|
P = 0.49
|
KIM-1 [pg/ml]
|
436.72 (±78.79)
|
388.17 (±42.29)
|
405.54 (±59.65)
|
P = 0.32
|
Abbreviations: L group – TMA low-dose group; H group – TMA high-dose group. All data are expressed as the mean ± SD.
FMOs gene and protein expression
The FMO3 gene expression in the renal cortex and renal medulla was significantly lower in the L group than in the control group. The L group had significantly higher FMO1 gene expression in the renal medulla than the control group. Additionally, the H group had significantly lower FMO3 gene expression in the renal cortex than the control group. There were no significant differences between groups in the FMO5 gene expression in evaluated tissues (Fig. 3).
Figure 3 title: FMOs genes expression
Figure 3. FMOs in the kidney, liver and lungs. RT-qPCR analysis of FMO1, FMO3 and FMO5 transcript levels in the (a) renal cortex, (b) renal medulla, (c) liver and (d) lungs in rats maintained either on tap water (control group) or low and high dose of TMA. Abbreviations: TMA, trimethylamine; FMO, Flavin-containing monooxygenase. All data are expressed as the median, Q1, Q3, MIN, MAX (n = 6; use arbitrary units); Kruskal-Wallis test followed by post-hoc Dunn’s test. *P < 0.05 vs. tap water group; **P < 0.01 vs. tap water group.
Renin-angiotensin system gene expression
The AGT gene expression in the renal medulla was significantly lower in the L group than in the control group. The AGTr1b gene expression in the renal cortex was increased in the L group compared to the control group. There were no significant differences between groups in the REN, AGTr1a, and AGTr2 gene expression (Additional Fig.1).
Histopathological evaluation
The control group showed no pathological changes in the liver or heart tissue. Nevertheless, in the kidneys of control animals, we observed some lymphohistiocytic infiltrates in the stroma, Bowman's capsule hyperplasia and thickening of the basal membranes of the tubules and vessels of the glomerulus.
In the kidneys of the L and H groups, the severity of the abovementioned changes was greater. Specifically, in the L and H groups, the pronounced thickening of the membranes of the substate tubules and glomerular vessels, mild degeneration of renal bodies with glomerulosclerosis, the hypertrophy of the tunica media of arteries and arterioles with vacuolization and degeneration of smooth myocytes were present. Moreover, mild to moderate chronic progressive nephropathy (CPN) can be described in rats from L and H groups. These changes are more pronounced in the H group (Fig. 4).
No pathological changes were found in the liver of the L group. Rats from the H group showed slight to moderate hepatocyte edema and cytoplasmic vacuolization, indicative of hydropic degeneration with features of acid degeneration. Moreover, there is marginalization of chromatin in the hepatocytes’ nuclei as well as prominent large 1-3 nucleoli. In the control and L groups, no significant lesions in the heart were found. In the H group in the myocardium, a focal wave course of cardiomyocytes, reduced visibility of cytoplasm striation and slight vacuolation of the cytoplasm were observed (Fig. 5).
Figure 4 title. Histopathology of the kidney.
Figure 4. Histopathology of the kidney. (a) Renal convoluted tubules (original magnification 10X, H&E stain), (b) renal medium size artery (original magnification 10X, H&E stain), (c) renal medium size artery (original magnification 40X, H&E stain) in rats maintained either on tap water (control group) or low and high dose of TMA. Legend: TMA, trimethylamine; L – lumen of convoluted renal tubules; Black arrow – vacuoles of various sizes within the cell cytoplasm; White arrow – endothelium.
Figure 5 title. Histopathology of the heart.
Figure 5. Histopathology of the heart. (a) Myocardium of left ventricle (original magnification 10X, H&E stain), (b) myocardium of left ventricle (original magnification 40X, H&E stain) in rats maintained either on tap water (control group) or low and high dose of TMA. Legend: TMA, trimethylamine.