Tryptophan metabolites suppress the Wnt pathway and promote adverse limb events in chronic kidney disease
Nkiruka V. Arinze, … , Nader Rahimi, Vipul C. Chitalia
Nkiruka V. Arinze, … , Nader Rahimi, Vipul C. Chitalia
Published November 9, 2021
Citation Information: J Clin Invest. 2022;132(1):e142260. https://doi.org/10.1172/JCI142260.
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Research Article Nephrology Vascular biology

Tryptophan metabolites suppress the Wnt pathway and promote adverse limb events in chronic kidney disease

Abstract

Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress β-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated β-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute–specific mouse models showed diminished β-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma’s AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/β-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.

Authors

Nkiruka V. Arinze, Wenqing Yin, Saran Lotfollahzadeh, Marc Arthur Napoleon, Sean Richards, Joshua A. Walker, Mostafa Belghasem, Jonathan D. Ravid, Mohamed Hassan Kamel, Stephen A. Whelan, Norman Lee, Jeffrey J. Siracuse, Stephan Anderson, Alik Farber, David Sherr, Jean Francis, Naomi M. Hamburg, Nader Rahimi, Vipul C. Chitalia

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Figure 3

IS downregulates β-catenin dependent on S33 residue in the degron motif.

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IS downregulates β-catenin dependent on S33 residue in the degron motif....
(A) β-Catenin N terminus contains a degron motif that controls its degradation. Black asterisk marks β-catenin S33A. Myc-tagged truncations lack N terminus (delN) and C terminus (delC). (B) ECs pretransfected with Myc-tagged β-catenin WT or S33A were treated with IS. Representative images of 3 independent experiments. (C) ECs pretransfected with Myc-tagged delC or delN β-catenin were treated with IS. Representative images of 3 independent experiments. (D) ECs transfected with WT β-catenin were treated with IS (50 μM) and cycloheximide (30 μg/mL) for indicated times. Average of normalized β-catenin from 4 independent experiments is shown. Error bars show SD. Independent Student’s t tests were performed at different time points. **P = 0.01; *P = 0.04. (E) Half-life study of Myc-tagged β-catenin S33A was performed as above from 4 independent experiments. Error bars show SD. (F) ECs pretransfected with Myc-tag β-catenin WT or S33A were treated with 50 μM IS and 10 μM MG132 before harvest. Immunoprecipitation was performed. Lysates were probed separately with anti-Myc-tag antibody. Representative images of 3 independent experiments are shown. (G) ECs stably expressing LS and Fu LS constructs were cotransfected with Myc-tagged WT or S33A β-catenin and were treated with IS or DMSO followed by a luciferase assay. Average of 3 experiments done in triplicate is shown. Error bars show SD. Independent Student’s t tests were performed. *P = 0.043; **P = 0.003; #P <0.0001. (H) ECs stably expressing LS and FuLS constructs were cotransfected with Myc-tagged WT and increasing amounts of S33A β-catenin (shown as + and ++ marks). Expression of β-catenin WT or S33A was confirmed (Supplemental Figure 5E). Average of 3 experiments done in triplicate is shown. Error bars show SD. Independent Student’s t tests were performed. **P = 0.002; #P = 0.001.