URAT1 is expressed in cardiomyocytes and dotinurad attenuates the development of diet-induced metabolic heart disease

Abstract

We recently reported that the selective inhibition of urate transporter-1 (URAT1), which is primarily expressed in the kidneys, ameliorates insulin resistance by attenuating hepatic steatosis and improving brown adipose tissue function in diet-induced obesity. In this study, we evaluated the effects of dotinurad, a URAT1-selective inhibitor, on the hearts of high-fat diet (HFD)-fed obese mice for 16-20 weeks and on neonatal rat cardiomyocytes (NRCMs) exposed to palmitic acid. Outside the kidneys, URAT1 was also expressed in cardiomyocytes and indeed worked as a uric acid transporter. Dotinurad substantially attenuated HFD-induced cardiac fibrosis, inflammatory responses, and cardiac dysfunction. Intriguingly, among various factors related to the pathophysiology of diet-induced obesity, palmitic acid significantly increased URAT1 expression in NRCMs and subsequently induced apoptosis, oxidative stress, and inflammatory responses via MAPK pathway, all of which were reduced by dotinurad. These results indicate that URAT1 is a potential therapeutic target for metabolic heart disease.

Keywords: Cell biology; Pathophysiology; Physiology.

Graphical abstract

Figure 1

URAT1 is expressed in the murine heart (A) URAT1 mRNA expression in hearts and kidneys obtained from NFD mice, as assessed by qRT-PCR (n = 3 each). (B) The protein expression of URAT1 in hearts and kidneys obtained from NFD mice (n = 3 each). (C) Immunohistochemical analyses of URAT1 expression in kidney cortical sections (upper panels) and hearts (middle panels) in NFD mice. URAT1-positive cells in cardiac vascular endothelial cells (white arrow) and cardiomyocytes are shown in the lower panels. The statistical analysis was performed using a two-tailed Student’s t test. Data represent the mean ± SEM. ∗∗∗p < 0.001 and ∗p < 0.05 between the indicated groups. Black arrows indicate proximal convoluted tubules, and yellow arrows indicate distal convoluted tubules.

Figure 2

URAT1-selective inhibitor treatment reduces cardiac fibrosis and inflammatory cytokines in HFD-fed mice (A) The heart weight at 4 weeks after treatment with or without dotinurad (NFD, n = 11; NFD+Doti, n = 11; HFD, n = 14; HFD+Doti, n = 12). (B) Representative hematoxylin-eosin staining of cardiac tissues. (C) Representative micrographs of histochemical staining of connective tissues using Masson’s trichrome. (D) The area of fibrosis assessed by Masson’s trichrome staining (NFD, n = 3; NFD+Doti, n = 3; HFD, n = 4; HFD+Doti, n = 4). (E–H) URAT1 protein expression in NFD and HFD mice with or without dotinurad treatment (NFD, n = 6; NFD+Doti, n = 6; HFD, n = 8; HFD+Doti, n = 6). The relative mRNA expression levels of TNF-α (F), MCP1 (G), and IL-1β (H) in heart tissue ([TNFα] NFD, n = 7; NFD+Doti, n = 6; HFD, n = 9; HFD+Doti, n = 8; [MCP1] NFD, n = 10; NFD+Doti, n = 6; HFD, n = 13; HFD+Doti, n = 8; [IL-1β] NFD, n = 8; NFD+Doti, n = 6; HFD, n = 9; HFD+Doti, n = 9). The statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Data represent the mean ± SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 between the indicated groups. HE, hematoxylin-eosin, HW, heart weight; TL, tail length; NFD, normal fat diet; HFD, high-fat diet; Doti, dotinurad.

Figure 3

URAT1-selective inhibitor treatment ameliorates HFD-induced cardiac dysfunction as assessed by echocardiography (A) Representative M-mode echocardiograms obtained from NFD or HFD mice with or without dotinurad. (B–G) The data of the indicated echocardiographic parameters are shown (NFD, n = 11; NFD+Doti, n = 11; HFD, n = 13; HFD+Doti, n = 14). The statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Data represent the mean ± SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 between the indicated groups. Doti, dotinurad; EF, ejection fraction; FS, fractional shortening; HFD, high-fat diet; IVSd, diastolic thickness of intraventricular septum; LVDd, left ventricular end-diastolic dimension; LVDs, left ventricular end systolic dimension; LVPWd, left ventricular posterior wall thickness; NFD, normal fat diet.

Figure 4

Cardiomyocytes take up UA via URAT1 (A) A schematic diagram of the experimental protocol. (B) The intracellular UA levels in NRCMs exposed to the indicated treatments for 60 min (n = 5 each). The statistical analysis was performed using a two-tailed Student’s t test or Mann‒Whitney U test. Data represent the mean ± SEM. ∗∗p < 0.01 and ∗p < 0.05 between the indicated groups. UA, uric acid.

Figure 5

PA induced the expression of URAT1 in NRCMs (A–C) A schematic diagram of the experimental protocol. URAT1 mRNA (n = 6 each) (B) and protein expression (n = 5 each) (C) in NRCMs exposed to the indicated treatments for 24 h. (D) Representative immunoblots and quantitative analyses of URAT1 in NRCMs transfected with URAT1 siRNA (siURAT1) or vehicle control siRNA (siNC) exposed to the indicated treatments for 24 h. (E) Immunofluorescence analyses of URAT1 expression in NRCMs transfected with siURAT1 or siNC. Cells were stained with URAT1 (red), Troponin-T (Green), and DAPI (blue). The statistical analysis was performed using a two-tailed Student’s t test or Mann‒Whitney U test in (B–D). Data represent the mean ± SEM. ∗∗p < 0.01 and ∗p < 0.05 between the indicated groups. PA, palmitate; Doti, dotinurad.

Figure 6

URAT1-selective inhibitor alleviates PA-induced MAPK phosphorylation, apoptosis, ROS, and inflammation in NRCMs (A) Representative immunoblots (upper) and quantitative analyses (lower) of p-p38, p38, p-ERK, ERK, p-JNK, and JNK from NRCMs exposed to the indicated treatments for 24 h ([p-p38/p38] and [p-ERK/ERK] n = 4 each; [p-JNK/JNK] n = 5 each). (B) Representative immunoblots (upper) and quantitative analyses (lower) of p-p38, p38, p-ERK, ERK, p-JNK, and JNK from NRCMs transfected with URAT1 siRNA (siURAT1) or vehicle control siRNA (siNC) exposed to the indicated treatments for 24 h (n = 4 each). (C) Representative immunoblots and quantitative analyses of the indicated apoptosis markers from NRCMs exposed to the indicated treatments for 24 h ([c-caspase 3] and [c-caspase 9] n = 6 each; [Bax] n = 4 each). (D) XO activity (H₂O₂ production using xanthine as a substrate) in NRCMs exposed to the indicated treatments for 24 h (n = 5 each). (E) The intracellular UA levels in NRCMs exposed to the indicated treatments for 24 h (n = 6 each). (F) The relative mRNA expression of the indicated inflammatory markers in NRCMs exposed to the indicated treatments for 24 h (n = 4 each). The statistical analysis was performed using a Mann‒Whitney U test in (A, B, C, and F) or a one-way ANOVA followed by Tukey’s post hoc test in (D and E). Data represent the mean ± SEM. ∗∗p < 0.01 and ∗p < 0.05 between the indicated groups. ROS, reactive oxygen species; PA, palmitate; Doti, dotinurad; XO, xanthine oxidase; UA, uric acid.