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Tubular β-catenin and FoxO3 interactions protect in chronic kidney disease
Stellor Nlandu-Khodo, … , Ethan Lee, Leslie S. Gewin
Stellor Nlandu-Khodo, … , Ethan Lee, Leslie S. Gewin
Published May 5, 2020
Citation Information: JCI Insight. 2020;5(10):e135454. https://doi.org/10.1172/jci.insight.135454.
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Research Article Cell biology Nephrology

Tubular β-catenin and FoxO3 interactions protect in chronic kidney disease

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Abstract

The Wnt/β-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. β-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/β-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular β-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) β-catenin specifically in murine proximal tubules. Mice with increased tubular β-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor–dependent β-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular β-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of β-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about β-catenin signaling and CKD by showing a protective effect of proximal tubule β-catenin in CKD and identified a potentially new transcriptional target of β-catenin/FoxO3 signaling that has therapeutic potential for CKD.

Authors

Stellor Nlandu-Khodo, Yosuke Osaki, Lauren Scarfe, Haichun Yang, Melanie Phillips-Mignemi, Jane Tonello, Kenyi Saito-Diaz, Surekha Neelisetty, Alla Ivanova, Tessa Huffstater, Robert McMahon, M. Mark Taketo, Mark deCaestecker, Balakuntalam Kasinath, Raymond C. Harris, Ethan Lee, Leslie S. Gewin

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Figure 3

Oxidative stress reduces β-catenin/LEF/TCF-dependent signaling and augments β-catenin/FoxO signaling.

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Oxidative stress reduces β-catenin/LEF/TCF-dependent signaling and augme...
(A) PT cells were treated with varying doses of Wnt3a either in control conditions (PT medium as described in Methods) or with oxidative stress (H2O2 100 μM in serum-free DMEM/F12) for 16 hours, and Axin2 transcripts were measured by qPCR and normalized to Gapdh. (B) PT cells stably transfected with a Topflash reporter construct (see Methods) were treated with various doses of Wnt3a in either control or oxidative stress medium (H2O2 100 μM in serum-free DMEM/F12). A luminometer measured the TCF/LEF-dependent activity (Steady Glo), which was normalized to cell number by using Cell Titer assay. For both A and B, Holm-Šídák multiple-comparisons test was used. (C) Cells treated with AA for 5 days showed increased oxidative stress reflected by increased nitrotyrosine on immunoblots. PT cells were treated with AA (30 μM), Wnt3a (10 ng/mL) was added during the last 48 hours, and nuclei were isolated (see Methods) and immunoblotted for FoxO1, FoxO3, or histone H3 for loading (D), and the results from 3 separate experiments were quantified (E). (F) PT cells were treated ± Wnt3a (10 ng/mL) and oxidative stress (H2O2 100 μM) for 16 hours, and then nuclei were isolated and coimmunoprecipitation was performed. Nuclear isolates had either β-catenin pull-down or IgG control, then were immunoblotted with FoxO3, and histone H3 was used for loading control of nuclear input. The nuclear input was also immunoblotted with α-tubulin, to assess for nuclear purity, and β-catenin. Levels of FoxO3 from the coimmunoprecipitation, normalized to histone H3 (nuclear input), were quantified from 3 separate experiments (G), as were nuclear β-catenin levels (H). Student’s t test was used for statistical analyses in G and H, and ANOVA was used for multiple comparisons in E with *P < 0.05 and **P < 0.01.

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