Abstract
Background and Aims
In chronic kidney disease (CKD), impaired kidney function results in the accumulation of uremic toxins, which exert deleterious biological effects, contributing to cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as p-cresyl sulfate, indoxyl sulfate and indole-3-acetic acid (IAA), originate from phenolic and indolic compounds, which are end products of the gut bacterial metabolization of aromatic amino acids (AAA). This study investigated the microbial composition in different stages of CKD by isolating, identifying and quantifying PBUT precursor-generating bacteria from fecal samples.
Method
Using fecal samples from patients in CKD stage 1 (n=6) and stage 5 (n=6), bacteria were cultured in a yeast casitone fatty acid glucose broth medium supplemented with AAA under aerobic (2d at 37°C) and anaerobic conditions (7d at 37°C), and confirmed as PBUT precursor-generating bacteria based on their generation capacity of phenolic and indolic compounds, measured with (U)HPLC.
Next, fecal DNA from 14 controls, 111 non-dialyzed and 27 dialyzed CKD patients was used to quantify the total bacterial number but also of 11 of the identified PBUT precursor-generating bacteria with qPCR. Using a Kruskal-Wallis test, bacterial loads were compared between the different CKD stages and control. Correlations between disease stages (control and CKD 1-5) and the abundance of bacterial species were assessed with the Spearman’s rank test.
Results
In total, 150 different bacterial species were isolated from the CKD fecal samples, of which 101 were identified and 92 classified as PBUT precursor-generating bacteria. In general, p-cresol and phenol were mainly generated under anaerobic conditions, while indole and IAA were generated under both aerobic and anaerobic conditions. Phenolic compounds and IAA were predominantly generated by bacterial species belonging to the Bacteroidaceae, Clostridiaceae, Enterococcaceae and Tannerellaceae, while indolic compounds were mainly generated by Bifidobacteriaceae and Enterobacteriaceae.
Quantitative analysis of 11 confirmed PBUT precursor-generating bacteria revealed a higher abundance of Streptococcus spp. and Enterobacteriaceae in fecal samples from HD patients compared to controls and early CKD stages, and for Roseburia spp. compared to CKD 5. Moreover, in HD, the abundance of Clostridioides difficile and Lactobacillus spp. was increased compared to CKD 1-5, and of Escherichia coli compared to control (all p>0.05). The abundance of Bacteroides spp., Faecalibacterium prausnitzii, Akkermansia muciniphila and Bifidobacterium spp. as well as the total number of bacteria was comparable among the different CKD stages and controls. Finally, decrease in kidney function (ranging from control to CKD 5) positively correlated with the abundance of Enterobacteriaceae (rs=0.210), and E. coli (rs=0.286), while an inverse correlation was found with Streptococcus spp. (rs=-0.255), Butyricoccus spp. (rs=-0.326), F. prausnitzii (rs=-0.250), Roseburia spp. (rs=-0.342) and Bifidobacterium spp. (rs=-0.303) (all p>0.05).
Conclusion
The identified PBUT precursor-generating bacteria are potential targets to reduce the plasma PBUT levels in CKD. In addition, in this CKD cohort, based on qPCR, an altered gut microbial composition with the progression of CKD could be established/confirmed.
Isolation and Quantification of Uremic Toxin Precursor-Generating Gut Bacteria in Chronic Kidney Disease Patients
