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 2

IS augments polyubiquitination and degradation of β-catenin in ECs.

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IS augments polyubiquitination and degradation of β-catenin in ECs. (A)....
(A). qRT-PCR analysis of ECs treated with IS 50 μM or DMSO (control) for different times was performed. The average cycle threshold (Ct) values performed in triplicate are shown. Error bars show SD. (B). ECs were treated with IS or vehicle (Veh) (DMSO) for 24 hours and cycloheximide (30 μg/mL) for the indicated time. Representative images of 4 independent experiments are shown. (C) β-Catenin was normalized to actin and represented as the percentage of remaining β-catenin at each time point. Average of normalized β-catenin from 4 independent experiments is shown. Error bars show SD. Independent Student’s t tests were performed. IS treatment at each time point compared with that of vehicle-treated cells. *P = 0.03; **P = 0.001. (D) ECs pretreated with 5% control human serum and spiked with IS corresponding to the different human CKD stages. Cells were exposed to 10 μM MG132 overnight. Immunoprecipitation was performed. The blot was reprobed with anti–β-catenin antibody. Representative images of 3 independent experiments are shown. (E) Ubiquitinated β-catenin was normalized to immunoprecipitated β-catenin. Average of normalized ubiquitinated β-catenin from 3 experiments is shown. Student’s t test with Bonferroni’s correction was performed for multiple comparisons. Error bars show SD. **P = 0.01; ***P < 0.001, compared with vehicle-treated cells. Blue dotted lines show IS levels corresponding to different CKD stages (Supplemental Table 2). (F) ECs pretreated with pooled control or uremic sera were processed as above. Blot was reprobed for β-catenin. Representative images of 3 independent experiments are shown. (G) Average of normalized ubiquitinated β-catenin from 3 experiments is shown. Independent Student’s t tests were performed. Error bars show SD. ***P < 0.001.