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 1

Uremic serum and IS downregulate Wnt/β-catenin signaling in ECs.

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Uremic serum and IS downregulate Wnt/β-catenin signaling in ECs. (A). Pr...
(A). Primary human microvascular ECs exposed to 5% pooled uremic or control sera. Equal amounts of protein were probed for β-catenin and separately for active β-catenin. Actin served as a loading control. Representative images from 3 independent experiments each done in duplicate are shown. (B) Representative images of 3 independent experiments of ECs treated with water-soluble uremic toxins. Con, control; Creat, creatinine; Oxalic, oxalic acid; Homo, homocysteine. (C). ECs pretreated with 5% of control serum and spiked with IS underwent fractionation. Tubulin and fibrillarin served as markers of fractions and loading controls. Representative images from 4 independent experiments. (D) β-Catenin was normalized to loading controls for their fractions. Average of 4 independent experiments is shown. Error bars show SD. The cytosol and nuclear fractions were analyzed separately and compared with the control (IS = 0 μM) using Student’s t test with Bonferroni’s correction of multiple comparisons. ***P < 0.001. (E) Serum-starved ECs expressing LS were treated with IS-spiked control human serum. Scatter plot of luciferase activity from 2 independent experiments done in quadruplet is shown. The horizontal line in each group corresponds to the median. The blue dotted line corresponds to IS levels in different CKD stages (Supplemental Table 2). A linear regression was performed. (F) Luciferase activity assay was performed as above in ECs treated with Wnt3a and IS spiked control human sera. A scatter plot of 2 independent experiments done in quadruplet is shown. A linear regression was performed. (G) Serum-starved ECs pretreated with vehicle (PBS + 1% BSA) or Wnt3a (20 ng/mL medium) with IS (50 μM) were stained. Representative images of 100 ECs/group. Profile plots were generated. Note different y axis scales between IS-treated and control samples. Scale bars: 5 μm. (H) Fifty ECs/group were analyzed. The dot plot represents the integrated density of nuclear β-catenin normalized to the surface area of nucleus. The line corresponds to the median. Independent Student’s t tests were performed to compare the groups.***P = 0.003; ****P < 0.001.