Alteration of Gut Microbiome and Correlated Amino Acid Metabolism Contribute to Hyperuricemia and Th17-Driven Inflammation in Uox-KO Mice

Although gut dysbiosis had been demonstrated to be an important factor affecting hyperuricemia (HUA) and gout, little is known for its potential mechanistic connections. In this study, Uox-KO mice model that with spontaneously developed pronounced HUA and urate nephropathy was used to explore the pathophysiologic mechanism of microbiota alterations in HUA and gout with integrated multi-omics analysis. 16S rRNA gene sequencing was performed to characterize the characteristic bacteria, and untargeted LC/MS analysis was applied to reveal the featured metabolites. Our results showed there was a significant shift in gut microbiota composition and function in Uox-KO mice compared to WT mice and apparent metabolomics differences between the two groups. Among them, amino acids metabolism appears to play a critical role. Correlation analysis further revealed that the characteristic metabolites were strongly influenced by the discrepant bacterial genera. Furthermore, impairment of intestinal integrity and profound alterations in the profile of solute carrier family resulted in dysregulation of amino acids transportation, which subsequently impacted serum uric acid level and CD4+ Th17 driven inflammation. Together, these data indicate that gut dysbiosis promotes purine metabolism disorder and inflammation in Uox-KO mice. Remodeling the gut microbiota is a promising strategy to combat HUA and gout.


Supplementary procedure for LC-MS analysis
The chromatographic separation was performed at 40 ℃ with a column of BEH C18 (100 mm × 2.1 mm, 1.7 µm, Waters, Milford, MA, USA). The two mobile phases consisted of 0.1 % formic acid aqueous solution (solvent A) and acetonitrile/ isopropanol (v/v, 1:1) with 0.1 % formic acid (solvent B). The flow rate was 0.40 mL/min and mobile phase (A: B) elution gradient was as follows: 95 %: 5 % for 0 min, 80%: 20% for 0 to 3.0 min, 5 %: 95 % for 3.0 to 9.0 min, 5 %: 95% for 9.0 to 13.0 min, 95 %: 5 % for 13.0 to 13.1 min, and 95 %: 5 % for 13.1 to 16.0 min. As a part of the system conditioning and quality control process, a pooled quality control sample (QC) was prepared by mixing equal volumes of all samples. The QC samples were disposed and tested in the same manner as the analytic samples.

Supplementary procedure for Western blotting analysis
Equal mass of intestinal proteins (30 μg/lane) were used for electrophoresis in a 10% or 8% SDS-PAGE gel. Protein bands in the gel of SDS-PAGE were electrophoretically transferred onto PVDF membrane. After blocking with 5 % milk in TBST buffer for 1 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4 ℃ with shaking. The membranes were then washed 3 times with TBST and incubated with secondary antibody for 1 h at room temperature.

Figure S2
(A) The relative abundance of the strains that positively correlated to SUA in co-housed WT mice. "ns" represents not significant; * p < 0.05；** p < 0.01.

(B)
The relative abundance of kidney amino acids (N = 7 mice/group). "ns" represents not significant.
(C) Spearman's rank correlation analysis between discrepant microbial taxa and serum amino acids. Positive correlations are displayed in red and negative correlations in blue. The intensity of the color is proportional to the correlation coefficient. + p < 0.05; # p < 0.01.
(D) Spearman's rank correlation analysis between discrepant microbial taxa and kidney amino acids. + p < 0.05; # p < 0.01. Figure S4 (A) Pairwise comparison of discrepant microbial taxa is shown with a color gradient signifying Spearman's correlation coefficient. Diamonds size corresponds to the value of the correlation coefficients. Diamonds without a number denote the statistically non-significant pairwise correlations. Gouty symptoms, CD4 + T cells and metabolite functional composition were related to discrepant microbial taxa by Mantel tests. Line width corresponds to the Mantel's r statistic for the corresponding distance correlations, and line color denotes the statistical significance based on 999 permutations. * p < 0.05; ** p < 0.01; *** p < 0.001.