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. 2021 May 14;9(6):1716-1726.
doi: 10.1016/j.gendis.2021.04.005. eCollection 2022 Nov.

Tetratricopeptide repeat domain 36 deficiency mitigates renal tubular injury by inhibiting TGF-β1-induced epithelial-mesenchymal transition in a mouse model of chronic kidney disease

Xin Yan  1 , Rui Peng  1 , Yilu Ni  1 , Lei Chen  1 , Qingling He  1 , Qianyin Li  1 , Qin Zhou  1
Affiliations

Tetratricopeptide repeat domain 36 deficiency mitigates renal tubular injury by inhibiting TGF-β1-induced epithelial-mesenchymal transition in a mouse model of chronic kidney disease

Xin Yan et al. Genes Dis. .

Abstract

The damage of proximal tubular epithelial cells (PTECs) is considered a central event in the pathogenesis of chronic kidney disease (CKD) and deregulated repair processes of PTECs result in epithelial-mesenchymal transition (EMT), which in turn aggravates tubular injury and kidney fibrosis. In this study, we firstly revealed that the reduction of TTC36 is associated with unilateral ureteral obstruction (UUO)-induced CKD; besides, ablation of TTC36 attenuated tubular injury and subsequent EMT in UUO-treated mice kidneys. Consistently, TTC36 overexpression promoted EMT in TGF-β1-induced HK2 cells. Moreover, TTC36 elevated the protein expression of CEBPB, which was involved in the regulation of TGF-β/SMAD3 signaling, and augmented SMAD3 signaling and downstream genetic response were reduced by CEBPB silencing. Collectively, our results uncovered that TTC36 deficiency plays a protective role in tubular injury and renal fibrosis triggered by UUO; further, TTC36 overexpression exacerbated TGF-β/SMAD3 signaling via elevating the stability of SMAD3 and CEBPB, suggesting that TTC36 inhibition may be a potential strategy in the therapy of obstructive nephropathy.

Keywords: CCAAT enhancer binding protein beta; Chronic kidney disease; Epithelial−mesenchymal transition; Renal fibrosis; SMAD family bember 3; Tetratricopeptide repeat domain 36.

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Figures

Fig. 1
Figure 1
The reduction of TTC36 in kidneys was associated with CKD. (A) Surgery strategy of UUO to induce CKD. (B) Representative images of IHC staining for TTC36 in kidney sections from UUO-induced WT mice for 7 days, Sham group as a control (n = 3; upper images scale bars, 100 μm; lower images scale bars, 25 μm). (C) Western blotting for TTC36 and VIM in the kidneys of mice with UUO-induced CKD (3, 7 and 14 days after UUO-treatment; n = 3 per group); β-ACTIN was used as a loading control. (D) Quantitative analysis for the expression of TTC36 relate to β-ACTIN by detecting integral optical density for (C). (E) Quantitative analysis for the expression of VIM relate to β-ACTIN using the detection of integral optical density for (C). Data are shown as means ± SD. Statistically significant differences were calculated by Student's t-test and ANOVA. ∗P < 0.05 versus sham group. Results are representative of three independent experiments. UUO, unilateral ureteral obstruction; CKD, chronic kidney disease; TTC36, tetratricopeptide repeat domain 36; VIM, Vimentin; SD, standard deviation; ANOVA, one-way analysis of variance.
Fig. 2
Figure 2
The absence of TTC36 mitigated UUO-mediated PTECs injury and inflammation. (A) Representative images of H&E staining in the kidneys of mice treated with UUO for 7 days, contralateral kidney as control (n = 3; upper images scale bars, 100 μm; lower images scale bars, 25 μm). (B) Tubular injury scores in mice were calculated. At least six random fields were taken from each kidney. (C) RT-qPCR analysis for Kim-1 and Ngal mRNA expression in the kidney of UUO-induced mice (WT-sham, Ttc36−/−-sham, WT-UUO, and Ttc36−/−-UUO; n = 3); 18s was used as an internal control. (D) RT-qPCR analysis for Il-1β, Il-6, and Tnf-α mRNA expression in the kidney of UUO-induced mice (WT-sham, Ttc36−/−-sham, WT-UUO, and Ttc36−/−-UUO; n = 3); 18s was used for an internal control. Data are shown as means ± SD. Statistically significant differences were determined by Student's t-test and one-way ANOVA. ##P < 0.01, and ###P < 0.001 versus WT-sham group; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus WT-UUO group. Results are representative of three independent experiments. H&E, hematoxylin-eosin; UUO, unilateral ureteral obstruction; RT-qPCR, quantitative real-time polymerase chain reaction; Kim-1, kidney injury molecule 1; Ngal, neutrophil gelatinase-associated lipocalin; Il-1β, interleukin-1β; Il-6, interleukin-6; Tnf-α, tumor necrosis factor; SD, standard deviation; ANOVA, one-way analysis of variance.
Fig. 3
Figure 3
TTC36 depletion ameliorated UUO-induced EMT and renal fibrosis. (A) Representative images of IHC staining for VIM in kidney sections from UUO-induced mice for 7 days, contralateral kidney as control (n = 3; upper images scale bars, 100 μm; lower images scale bars, 25 μm). (B) IHC scores in mice were analyzed. At least eight random fields were taken from each kidney. (C) Western blotting for CDH2, VIM, SNAI1, TWIST1, and TTC36 in the kidneys of mice with UUO-induced CKD (WT-UUO and Ttc36−/−-UUO; n = 3); β-Tubulin as a loading control. (D) The mRNA expression of Acta2, Vim, Fn1, and Col1a1 were detected by RT-qPCR (WT-sham, Ttc36−/−-sham, WT-UUO, and Ttc36−/−-UUO; n = 3); 18s was used as an internal control. (E) The mRNA expression of Snai1, Sani2, and Twist1 were detected by RT-qPCR (WT-sham, Ttc36−/−-sham, WT-UUO, and Ttc36−/−-UUO; n = 3); 18s was used as an internal control. Data are shown as means ± SD. Statistically significant differences were analyzed by Student's t-test and one-way ANOVA. ##P < 0.01 and ###P < 0.001 versus WT-contralateral group or WT-sham group; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus WT-UUO group. Results are representative of three independent experiments. IHC, immunohistochemical; VIM, Vimentin; CDH, cadherin 2; UUO, unilateral ureteral obstruction; CKD, chronic kidney disease; Fn1, fibronectin 1; Acta2, actin alpha 2 smooth muscle; Col1a1, collagen type I alpha 1 chain; RT-qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation; ANOVA, one-way analysis of variance.
Fig. 4
Figure 4
TTC36 overexpression promoted TGF-β/SMAD3 signaling and TGF-β1-induced EMT and cell cycle arrest. (A) Western blotting for SMAD2/3, p-SMAD3, and TTC36 in the kidneys of mice treated with UUO for 7 days; GAPDH as a loading control (WT-UUO and Ttc36−/−-UUO; n = 3). (B) Western blotting for SMAD3, p-SMAD3, and Flag in TGF-β1-induced HK2 cells for 48 h with or without TTC36 overexpression; β-Actin was used as a loading control. (C) Western blotting for CDH2, VIM, ACTA2, and SNAI1 in TGF-β1-induced HK2 cells with or without TTC36 overexpression; β-ACTIN was used as a loading control. (D) Representative images for flow cytometry analysis. TTC36 was overexpressed in HK2 cells followed by TGF-β1-treatment for 48 h. (E) The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle are shown. Data are shown as means ± SD. Statistically significant differences were analyzed by Student's t-test and one-way ANOVA. ##P < 0.01 versus Control group without TGF-β1-treatment; ∗∗P < 0.01 versus TGF-β1-induced control group. Results are representative of three independent experiments. p-SMAD3, phosphorylated SMAD3; CDH2, cadherin 2; UUO, unilateral ureteral obstruction; ACTA2, actin alpha 2, smooth muscle; GAPDH, glyceraldehyde-phosphate dehydrogenase; TGF-β1, transforming growth factor β1; HK2, human proximal tubular epithelial cell; SD, standard deviation; ANOVA, one-way analysis of variance.
Fig. 5
Figure 5
TTC36 promoted TGF-β/SMAD3 signaling via SMAD3 and CEBPB. (A) Western blotting for SMAD2/3, p-SMAD3, SNAI1, and Flag in TGF-β1-induced HK2 cells with or without TTC36 silenced; β-ACTIN was used as a loading control. (B) Proteins were extracted from HK2 cells and immunoprecipitation for Flag-marked TTC36 was performed with an anti-Flag antibody. Western blotting was performed by using the indicated antibodies. (C) HK2 cells expressing Flag-control or Flag-TTC36 were treated with 5 μg/ml cycloheximide for 0, 1, 2, or 3 h; Western blotting for SMAD3; β-ACTIN as a loading control (up); quantification of SMAD3 levels (relative to 0 h, below). (D) Western blotting for SMAD2/3, p-SMAD3, CEBPB, SNAI1, and Flag in TGF-β1-treated HK2 cells with or without CEBPB silenced; β-ACTIN was used as a loading control. (E) HK2 cells expressing Flag-control or Flag-TTC36 were treated with 5 μg/ml cycloheximide for 0, 1.5, 3, or 6 h; Western blotting for CEBPB; β-ACTIN as a loading control (up); quantification of CEBPB levels (relative to 0 h, below). (F) Western blotting for CEBPB and Flag in HK2 cells with or without TTC36 silence; β-ACTIN was used as a loading control. Data are exhibited as means ± SD. Statistically significant differences were analyzed by Student's t-test. ∗P < 0.05 and ∗∗P < 0.01 versus control group. Results are representative of three independent experiments. p-SMAD3, phosphorylated SMAD3; CEBPB, CCAAT enhancer binding protein β; TGF-β1, transforming growth factor β1; HK2, human proximal tubular epithelial cell; SD, standard deviation; CHX, cycloheximide.
Fig. 6
Figure 6
Schematic for TTC6 exacerbating TGF-β1-induced EMT via SMAD3 and CEBPB, exacerbating tubular injury in the mouse model of CKD.
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