Rifaximin stimulates nitrogen detoxification by PXR-independent mechanisms in human small intestinal organoids.

BACKGROUND AND AIMS
Recurrent hepatic encephalopathy (HE) is characterized by hyperammonemia in combination with neuropsychiatric abnormalities and is treated with lactulose and rifaximin. Rifaximin is a Pregnane X Receptor (PXR) agonist with low systemic and high intestinal bioavailability. The mechanisms by which it alleviates HE are unclear. We used human small intestinal (hSI) organoids to study whether rifaximin, via PXR activation, affects the epithelial biotransformation machinery, and to gain understanding of its low systemic availability.


METHODS
We generated PXR knockdown hSI organoids via lentiviral delivery of short hairpin RNAs. Organoids were cultured for 24 hours with rifaximin or rifampicin. RNA-sequencing and metabolomics were performed to analyse gene expression and amino acid metabolism. Luminal rifaximin was quantified by photospectrometry.


RESULTS
Treatment of wildtype hSI organoids with rifaximin resulted in >2-fold differential expression of 131 genes compared to DMSO. These effects were largely PXR-independent and related to amino acid metabolism. Rifaximin decreased expression of glutaminase-2 and increased expression of asparagine synthetase and solute carrier 7A11, thereby increasing intracellular glutamine and asparagine concentrations, indicating active ammonia detoxification. Rifaximin was apically excreted into the lumen in an ATP Binding Cassette B1 (ABCB1)-dependent manner.


CONCLUSIONS
Rifaximin - after uptake into enterocytes - stimulates intracellular nitrogen detoxification by PXR-independent mechanisms. Active apical excretion of rifaximin by ABCB1 into the intestinal lumen explains its low systemic bioavailability. Our study implies that rifaximin, next to modulation of the microbiome, has direct effects on ammonia scavenging in the human small intestinal epithelium.