Liquid chromatography coupled with high resolution mass spectrometry reveals the inhibitory effects of Huangkuisiwu formula on biosynthesis of protein-binding uremic toxins in rats with chronic kidney disease

Abstract

Chronic kidney disease (CKD) is recognized as a common disorder worldwide. Protein-binding uremic toxins that cannot be efficiently removed by extracorporeal renal replacement therapies, such as indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are associated with high risks of cardiovascular complications and high mortality in CKD population. This study aimed to explore the therapeutical effects of Huangkuisiwu formula (HKSWF) on CKD rats. Moreover, the underlying mechanisms of HKSWF to inhibit the biosynthesis of IS and PCS were studied. Untargeted metabolomics based on UHPLC-QTOF/MS was conducted to analyze the alterations of endogenous metabolites in plasma. Levels of IS and PCS in plasma and peripheral tissues, as well as levels of amino acids in colonic contents were analyzed by UHPLC-TQ/MS. Levels of indole and p-cresol, the precursors of IS and PCS, in feces and colonic contents were quantified by HPLC-FLD. mRNA and protein expression of sulfotransferase 1 a1 (SULT1A1) were determined by qPCR and Western blotting, respectively. The ability of colonic microbiota to metabolize amino acids into precursors, as well as the activity of sulfotransferase to catalyze precursors into uremic toxins were evaluated by detecting corresponding products from specific substrates. 16S rRNA sequencing were conducted to analyze the profile of gut microbiota. The results showed that HKSWF significantly alleviated the structural and functional impairment of kidney, as well as improved the global metabolic disorders in CKD rats. IS and PCS were identified as the key differential metabolites that contributed to the effects of HKSWF. HKSWF significantly reduced the levels of IS and PCS in plasma, kidney, liver and heart of CKD rats. HKSWF showed no significant effects on the expression of SULT1A1 or the activity of sulfotransferase. HKSWF significantly decreased the levels of indole and p-cresol in the colonic contents and feces of CKD rats, by inhibiting the ability of colonic microbiota to metabolize tryptophan and tyrosine into indole and p-cresol. Alterations in the profile of amino acids and gut microbiota in CKD rats were significantly improved by HKSWF treatment. Conclusively, HKSWF inhibited gut-microbiota mediated biosynthesis of indole and p-cresol, to alleviate the accumulation of IS and PCS in CKD rats.

Keywords: Chronic kidney disease; Gut microbiota; Huangkuisiwu formula; Indoxyl sulfate; Protein-binding uremic toxin; p-Cresyl sulfate.