Isolation and Quantification of Uremic Toxin Precursor-Generating Gut Bacteria in Chronic Kidney Disease Patients.

Tessa Gryp,Griet Glorieux,Marie Joossens,Geert R B Huys,Mario Vaneechoutte,Wim Van Biesen

doi:10.3390/ijms21061986

Abstract

In chronic kidney disease (CKD), impaired kidney function results in accumulation of uremic toxins, which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as p-cresyl sulfate, indoxyl sulfate and indole-3-acetic acid, originate from phenolic and indolic compounds, which are end products of gut bacterial metabolization of aromatic amino acids (AAA). This study investigates gut microbial composition at different CKD stages by isolating, identifying and quantifying PBUT precursor-generating bacteria. Fecal DNA extracts from 14 controls and 138 CKD patients were used to quantify total bacterial number and 11 bacterial taxa with qPCR. Moreover, isolated bacteria from CKD 1 and CKD 5 fecal samples were cultured in broth medium supplemented with AAA under aerobic and anaerobic conditions, and classified as PBUT precursor-generators based on their generation capacity of phenolic and indolic compounds, measured with U(H)PLC. In total, 148 different fecal bacterial species were isolated, of which 92 were PBUT precursor-generators. These bacterial species can be a potential target for reducing PBUT plasma levels in CKD. qPCR indicated lower abundance of short chain fatty acid-generating bacteria, Bifidobacterium spp. and Streptococcus spp., and higher Enterobacteriaceae and E. coli with impaired kidney function, confirming an altered gut microbial composition in CKD.

Highlights

In chronic kidney disease (CKD), impaired kidney function results in the accumulation of uremic toxins [1,2,3], which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality [4,5,6,7,8]
Well-known protein-bound uremic toxins (PBUTs), p-cresyl sulfate, p-cresyl glucuronide, indoxyl sulfate (IxS) and indole-3-acetic acid (IAA) originate in the colon, where aromatic amino acids (AAA) are metabolized by gut bacteria into phenolic (p-cresol and phenol) and indolic compounds [9,10,11,12]. p-Cresol and indole are sulfated in the colon mucosa and by the liver into pCS and IS, and a small part is glucuronidated into pCG [9,13,14], while IAA enters the circulation without further modifications [15,16,17]
Seven different media were compared based on the cultivation of a bacterial test panel consisting of 12 known uremic toxin precursor-generating bacterial species (Figure S1), and the cultivation of fecal bacteria from whole fecal samples (Table S1)

Summary

Introduction

In chronic kidney disease (CKD), impaired kidney function results in the accumulation of uremic toxins [1,2,3], which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality [4,5,6,7,8]. Ammonia is a bacteria-derived hydrolysis end product of urea, which accumulates in the circulation and enters the gut lumen via the entero-hepatic cycle [31] and by diffusion through the disrupted intestinal epithelial barrier as is typically seen in patients with CKD [32,33] This leads to a uremic milieu in the gut lumen, altering the gut microbial composition in favor of urease expressing bacteria in end stage kidney disease (ESKD) patients [27]. In a recent study by our group [34], the generation of fecal PBUT precursor metabolites by fecal bacteria did not alter with CKD progression nor compared to controls This suggests that the accumulation of PBUTs in plasma is mainly due to the impaired kidney function itself. The abundance of specific fecal PBUT precursor-generating bacteria in the different stages of CKD and in controls was investigated

Results

Quantification of Fecal Bacterial Species in Different Stages Of CKD

Study Population and Sample Collection

Culture Media

Quantification of Bacterial Species in Fecal Samples

Statistical Analysis