Renal fibrosis is induced by hyperactive Wnt/β-catenin pathway via microbial-mediated tryptophan metabolism-driven AhR signaling in rodents and humans

Renal fibrosis is a common pathological endpoint in progressive chronic kidney disease (CKD). Clinical evidence indicates that a decline in renal function is more closely associated with tubulointerstitial fibrosis (TIF) than with glomerular injury. Recent advances in multi-omics technologies have provided powerful tools for uncovering unrecognized disease molecular mechanisms. Metagenomic and metabolomic analyses were performed to profile the fecal microbiota and serum metabolites, respectively, and to identify tubulointerstitial damage (TID)-related bacterial taxa and metabolites. Identified serum metabolites were also determined in healthy controls and tubulointerstitial nephropathy (TIN) patients. The expression of aryl hydrocarbon receptor (AhR) and Wnt/β-catenin signaling–related genes and proteins was evaluated in obstructed kidney of unilateral ureteral obstruction (UUO) rats and AhR shRNA-treated UUO mice as well as in 1-hydroxypyrene (HP)-stimulated HK-2 cells untreated or treated with AhR shRNA. UUO induced progressive TID and TIF in rats. Alterations in gut microbiota composition, particularly changes in Enterocloster aldenensis (E. aldenensis) and Lactobacillus acidipiscis (L. acidipiscis), were strongly correlated with TID. In parallel, microbial-derived tryptophan catabolites (MDTCs), including tryptamine, indole-3-acetic acid (IAA), indole-3-lactic acid (ILA), indole-3-propionic acid (IPA), indole-3-acrylic acid, indole-3-aldehyde (IAld), and indoxyl sulfate were strongly associated with TID severity. Linear regression analyses revealed correlation coefficients exceeding 0.80 between E. aldenensis and IAA, ILA, and IPA, and between L. acidipiscis and IAld, indicating close relationships with progressive TIF. Similarly, the changes of 14 MDTCs were further demonstrated in TIN patients and they could separate TIN patients form healthy controls. Some MDTCs showed strongly correlation with estimated glomerular filtration rate in TIN patients and high values of area under the curve, sensitivity and specificity. These microbial and metabolic alterations were accompanied by activation of the AhR–Wnt/β-catenin signaling pathway. By contrast, AhR shRNA treatment inhibited mRNA expression of AhR and its downstream target genes, including cytochrome P450 family 1 subfamily A member 1 (CYP1A1), CYP1A2, CYP1B1 and cyclooxygenase-2 accompanied by suppressing nuclear AhR localization, retarded protein expression of Wnt1, β-catenin and Twist, enhanced E. aldenensis and L. acidipiscis abundances and reversed MDTC dysregulation in UUO mice. Bioactivity-directed isolation and identification demonstrated that polyporusterone A (PPA) from Polyporus umbellatus increased abundance of E. aldenensis and L. acidipiscis and normalized dysregulated MDTCs in UUO rats. PPA treatment suppressed intrarenal AhR signaling and Wnt1/β-catenin pathway. Consistent effects were observed in HP-induced HK-2 cells treated with PPA; however, AhR knockdown partially attenuated these inhibitory effects. Taken together, this study first demonstrated that the enrichment of pathogenic bacteria and depletion of probiotics-mediated dysregulation of MDTCs is closely linked to the activation of the AhR–Wnt/β-catenin signaling axis in UUO rat model. Targeting GM may represent a promising therapeutic strategy for CKD and renal fibrosis.