Short-Chain Fatty Acids Alleviate Hepatocyte Apoptosis Induced by Gut-Derived Protein-Bound Uremic Toxins.

Mingjuan Deng,Weiwei Li,Liang Zhao,Xiaoying Zhang,Xingqi Li,Jiahui Gong,Shaoyang Ge

doi:10.3389/fnut.2021.756730

Abstract

Chronic kidney disease (CKD) is characterized with the influx of uremic toxins, which impairs the gut microbiome by decreasing beneficial bacteria that produce short-chain fatty acids (SCFAs) and increasing harmful bacteria that produce gut-derived protein-bound uremic toxins (PBUTs). This study aimed to assess the proapoptotic effects of three major gut-derived PBUTs in hepatocytes, and the effects of SCFAs on apoptosis phenotype in vitro. HepG2 (human liver carcinoma cells) and THLE-2 (immortalized human normal liver cells) cell line were incubated with 0, 2, 20, 200, 2000 μM p-cresol sulfate (PCS), indoxyl sulfate (IS), and hippuric acid (HA), respectively, for 24 h. Flow cytometry analysis indicated that three uremic toxins induced varying degrees of apoptosis in hepatocytes and HA represented the highest efficacy. These phenotypes were further confirmed by western blot of apoptosis protein expression [Caspase-3, Caspase-9, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax)]. Human normal hepatocytes (THLE-2) are more sensitive to PBUTs-induced apoptosis compared with human hepatoma cells (HepG2). Mechanistically, extracellular HA could enter hepatocytes, increase reactive oxygen species (ROS) generation, and decrease mitochondrial membrane potential dose-dependently in THLE-2 cells. Notably, coculture with SCFAs (acetate, propionate, butyrate) for 24 h significantly improved HA-induced apoptosis in THLE-2 cells, and propionate (500 μM) represented the highest efficacy. Propionate reduction of apoptosis was associated with improving mitochondria dysfunction and oxidative stress in a manner involving reducing Caspase-3 expression, ROS production, and increasing the Bcl-2/Bax level. As such, our studies validated PBUTs accumulation might be an important cause of liver dysfunction in patients with CKD, and supplementation of SCFAs might be a viable way to protect the liver for patients with CKD.

Highlights

Chronic kidney disease (CKD) has become a serious health issue affecting 10–15% of the global population [1]
Uremic syndrome coupled with kidney dysfunction is characterized by retention of uremic toxins, and several of the most harmful uremic toxins are produced by gut bacteria, including hippuric acid (HA), indoxyl sulfate (IS), and p-cresol sulfate (PCS) [5]
PCS was found to induce oxidative stress, glutathione depletion, and cellular necrosis in a human liver cell line, more evidence is needed to clarify the effects of gut-derived protein-bound uremic toxins (PBUTs) on hepatocytes [11]

Summary

Introduction

Chronic kidney disease (CKD) has become a serious health issue affecting 10–15% of the global population [1]. The accumulation of PBUTs could exert harmful biological activity on other tissues or organs, mainly on the kidney and the cardiovascular system [8, 9]. IS and PCS could affect hepatic bile acid transport and mitochondrial functions [6, 8]. In this respect, Weigand et al revealed that HA mediated liver injury primarily via mitochondrial toxicity [12]. Apoptosis is critically involved in PBUTs-induced harmful biological activities. Lin et al found IS caused apoptosis via oxidative stress and mitogen-activated protein kinase (MAPK) signaling inhibition in human astrocytes [15]. Scarce research has been conducted to explore the proapoptotic effects of PBUTS on the liver, an extremely important organ to the metabolism, which attracted our scientific interest

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Methods

Results