Treatment with Gac Fruit Extract and Probiotics Reduces Serum Trimethylamine N-Oxide in Chronic Kidney Disease Rats

Abstract

Chronic kidney disease (CKD) affects more than 850 million people worldwide, contributing to morbidity and mortality, particularly through cardiovascular disease (CVD). The altered composition in CKD patients leads to increased production and absorption of uremic toxins such as trimethylamine (TMA) and its oxidized form, trimethylamine N-oxide (TMAO), which are associated with cardiovascular risks. This study investigated the potential of supplementary interventions with high-carotenoid-content gac fruit extract and probiotics to mitigate serum TMAO by modulating the gut microbiota. We conducted an animal study involving 48 male Wistar rats, divided into six groups: the control, CKD control, and four treatment groups receiving gac fruit extract, carotenoid extract, or combinations with Ligilactobacillus salivarius and Lactobacillus crispatus and Lactobacillus casei as a standard probiotic. CKD was induced in rats using cisplatin and they were supplemented with choline to enhance TMA production. The measures included serum creatinine, TMAO levels, gut microbiota composition, and the expression of fecal TMA lyase and intestinal zonula occluden-1 (ZO-1). CKD rats showed increased TMA production and elevated serum levels of TMAO. Treatment with gac fruit extract and selective probiotics significantly altered the composition of the gut microbiota by decreasing Actinobacteriota abundance and increasing the abundance of Bacteroides. This combination effectively promoted ZO-1 expression, reduced fecal TMA lyase, and subsequently lowered serum TMAO levels, demonstrating the therapeutic potential of these interventions. Our results highlight the benefits of gac fruit extract combined with probiotics for the effective reduction in serum TMAO levels in rats with CKD, supporting the further exploration of dietary and microbial interventions to improve outcomes in patients with CKD.

Keywords: TMAO; chronic kidney disease; gac fruit; gut microbiota; probiotics.

Figure 1

Body weight, heart weight and heart weight per body weight of experimental rats. The weight was measured at the end of the study. Values are presented as mean ± standard deviation (n = 8). * p < 0.05 vs. control. CKD: chronic kidney disease; GacCar: gac fruit extract supplemented; STDCar: standard carotenoid extract supplemented; GacPro: gac fruit extract with Ligilactobacillus salivarius and Lactobacillus crispatus supplemented; STDPro: lactobacillus casei supplemented.

Figure 2

Plasma uremic toxins and TMA lyase expression. (A) Plasma creatinine at week 15. (B) Comparison between serum TMAO at week 0 and week 15 in each experimental group. (C) Fecal TMA lyase RNA expression in week 15. Values are presented as mean ± standard deviation (n = 8). * p < 0.05; ** p < 0.01.

Figure 3

Intestinal zonula occludens type 1 (ZO-1) expression. (A) The immunohistochemistry of ZO-1 in the colon. (B) Comparison of the ZO-1 expression score between each group. 0: Expression of ZO-1 less than 10%; 1: 10–25% expression; 2: 26–50% expression; 3: 51–75% expression and 4: more than 75% expression. The ZO-1 expression scores are presented as the median with the interquartile range (n = 8). * p < 0.05 vs. CKD group. Arrow: immunohistochemical staining of the ZO-1 protein.

Figure 4

Fecal gut microbiome studies. The gut microbiome was determined using the 16sRNA sequencing method at the end of the study. (A) Shannon’s diversity index. (B) Richness. (C) Pielou’s evenness. (D) Bray–Curtis principal coordinate analysis (PCoA) of the fecal microbiota. Box and whisker plot showing the distribution of Shannon’s diversity index, richness, and Pielou’s evenness for each group. The boxes represent the interquartile range (IQR), with whiskers extending to the smallest and largest values.