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. 2022 Apr 6;27(7):2344.
doi: 10.3390/molecules27072344.

Rhein Improves Renal Fibrosis by Restoring Cpt1a-Mediated Fatty Acid Oxidation through SirT1/STAT3/twist1 Pathway

Xianrui Song  1 , Zesen Du  1 , Zhenqi Yao  1 , Xiaoyan Tang  1 , Mian Zhang  1
Affiliations

Rhein Improves Renal Fibrosis by Restoring Cpt1a-Mediated Fatty Acid Oxidation through SirT1/STAT3/twist1 Pathway

Xianrui Song et al. Molecules. .

Abstract

The latest progress in the field of renal fibrosis mainly focuses on the new concept of "partial epithelial-mesenchymal transition (pEMT)" to explain the contribution of renal tubular epithelial (RTE) cells to renal fibrosis and the crucial role of fatty acid oxidation (FAO) dysfunction in RTE cells for the development of renal fibrosis. FAO depression is considered to be secondary or occur simultaneously with pEMT. We explored the relationship between pEMT and FAO and the effect of rhein on them. Intragastric administration of rhein significantly improved the levels of BUN, Scr, α-SMA, collagen 1A and histopathological changes in UUO-rats. Transcriptomic and metabolomic analyses revealed that abnormal signaling pathways were involved in EMT and FAO disorders. RTE cell experiments showed that TGF-β could inhibit the activity of Cpt1a, resulting in ATP depletion and lipid deposition. Cpt1a inhibitor induced EMT, while Cpt1 substrate or rhein inhibited EMT, indicating that Cpt1a-mediated FAO dysfunction is essential for RTE cells EMT. Further studies showed that Cpt1a activity were regulated by SirT1/STAT3/Twist1 pathway. Rhein inhibits RTE cell EMT by promoting Cpt1a-mediated FAO through the SirT1/STAT3/Twist1 pathway. Surprisingly and importantly, our experiments showed that FAO depression occurs before EMT, and EMT is one of the results of FAO depression.

Keywords: Cpt1a-mediated fatty acid oxidation; SirT1/STAT3 pathway; Twist1; epithelial–mesenchymal transition; renal fibrosis; rhein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rhein attenuates UUO-induced renal fibrosis in rats. The left ureter of rat was ligated in two places and cut between the two ligatures (UUO operation). The rats were i.g. administered with or without rhein at 100 mg/kg per day from days 1 to 14 after UUO operation. (A) Structure of rhein; (B) renal coefficient; (C,D) contents of BUN and Scr in serum; (E) representative images of kidney samples stained with H&E and Masson (scale bars, 100 μm), and quantitative analysis of HE score and collagen area percentage; (F) representative images of kidney samples immunostained with α-SMA and Col1A (n = 3). Data were expressed as mean ± SD (n = 5). ## p < 0.01, ### p < 0.001, vs. sham rats; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. UUO rats.
Figure 2
Figure 2
Transcriptomic and metabolomic analysis of UUO-induced rat kidneys. Rats were operated and treated in the same manner as in Figure 1. Total RNA extracted from kidney samples was used for transcriptomic analysis, and acetonitrile extract from kidney samples was used for metabolomic analysis. (A) KEGG enrichment pathways of differential genes between UUO and sham rats with q-value < 0.05 and log2(FoldChange) > 1; RNA sequencing analysis of genes associated with (B) fibrosis (n = 4) and (C) fatty acid metabolism (n = 4); mRNA expressions associated with (D) fibrosis (Col1a1, Col3a1,Col4a1, TGF-β, Vim, Acta2, Fn1 and Twist1) and (E) fatty acid metabolism (Cpt1a, Cpt1b, Acot1, Decr1, Echs1 and Cpt2) in rat kidney samples were determined (n = 5); (F) heatmap of metabolites in rat kidney samples (n = 8); (G) KEGG enrichment pathways of differential metabolites between UUO and sham rats with VIP > 1 and p < 0.05; (H) network diagram of Twist1 gene, Cpt1a gene and differential metabolites in kidney of UUO and sham rats. Data were expressed as mean ± SD. ## p < 0.01, ### p < 0.001, vs. sham rats; * p < 0.05, ** p < 0.01, vs. UUO rats.
Figure 3
Figure 3
Rhein reverses TGF-β-induced EMT in RTE cells by promoting Cpt1a activity. (A) Cell viability in RTE cells incubated with rhein at indicated concentrations for 24 h. RTE cells were incubated with 10 ng/mL TGF-β and rhein at indicated concentrations for 24 h, (B) protein expressions of E-cadherin, α-SMA and Col1A; (C) representative cell images of lipid droplets stained with BODIPY (rhein at 50 μmol/L, ×630). (D) Intracellular ATP levels in RTE cells incubated with 10 ng/mL TGF-β and 50 μmol/L rhein, or with 40 μmol/L etomoxir, for 24 h. RTE cells were incubated with 10 ng/mL TGF-β and 10 or 50 μmol/L rhein for 24 h, (E) protein expression and (F) activity of Cpt1. RTE cells were incubated with 10 ng/mL TGF-β and 50 μmol/L rhein or 0.5 mmol/L carnitine, or with 40 μmol/L etomoxir, for 24 h, (G,I) mRNA expressions of E-cadherin, vimentin, Acta2 and Col1a1; (H) Cpt1 activity. RTE cells were incubated with 10 ng/mL TGF-β and 50 μmol/L for 24 h, (J) mRNA expressions of Cpt1a, Cpt1b and Cpt1c; (K) representative images of cells immunostained with Cpt1a (×630). Data were expressed as mean ± SD (n = 5). # p < 0.05, ## p < 0.01, ### p < 0.001, vs. blank cells; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. TGF-β- or etomoxir-induced cells.
Figure 4
Figure 4
Twist1 is essential for Cpt1a-mediated FAO depression in RTE cells. (A) mRNA and (B) protein expression of Twist1 in RTE cells incubated with 10 ng/mL TGF-β and 10 or 50 μmol/L rhein for 24 h. After Twist1 transfection, RTE cells were incubated with 10 ng/mL TGF-β and 50 μmol/L rhein for 24 h, (C) mRNA and (D) protein expressions of E-cadherin, vimentin, Acta2 and Col1a1, (E) Cpt1a mRNA and (F) Cpt1 activity were detected; (G,H) representative images of cells immunostained with Cpt1a or stained with BODIPY (×630); (I) intracellular ATP levels. Data were expressed as mean ± SD (n = 5). ## p < 0.01, ### p < 0.001, vs. blank cells; * p < 0.05, ** p < 0.01, vs. TGF-β-induced cells.
Figure 5
Figure 5
Rhein inhibits Twist1 expression by regulating SirT1/STAT3 pathway. (A) Protein expressions of Twist1, p-STAT3, E-cadherin, α-SMA and Col1A in RTE cells incubated with 10 ng/mL TGF-β and 50 μmol/L rhein or 2 μmol/L WP1066 for 24 h. (B) Protein expressions of Twist1 and p-STAT3 in RTE cells incubated with 10 ng/mL TGF-β after STAT3 transfection. (C) ChIP assay for STAT3 and Twist1 in RTE cells incubated with 10 ng/mL TGF-β and 50 μmol/L rhein for 24 h. RTE cells were incubated with 10 ng/mL TGF-β and 50 μmol/L rhein or 10 μmol/L resveratrol for 24 h, (D) protein expressions of SirT1 and p-STAT3; (E) protein expression of acetylated STAT3 (n = 4); (F) protein expressions of Twist1, E-cadherin, α-SMA and Col1A. After SirtT1 transfection, RTE cells were incubated with 10 ng/mL TGF-β and 50 μmol/L rhein for 24 h, (G) protein expressions of SirT1 and p-STAT3; (H) protein expression of acetylated STAT3 (n = 4). Data were expressed as mean ± SD (n = 5). # p < 0.05, ## p < 0.01, ### p < 0.001, vs. blank cells; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. TGF-β-induced cells.
Figure 6
Figure 6
Rhein promotes Cpt1a-mediated FAO via SirT1/STAT3/Twist1 pathway in UUO-induced rats. Rat UUO operation was in the same manner as in Figure 1. The rats were i.g. administered with or without rhein at 100 mg/kg or resveratrol at 20 mg/kg per day from days 1 to 14, (A) protein expressions E-cadherin, α-SMA and Col1A of kidney samples; (B,C) representative images of kidney samples stained with BODIPY and immunostained with Cpt1a and Twist1 (×630, n = 3); (D) mRNA expression of Cpt1a; (E) protein expressions for Twist1, SirT1, p-STAT3 and acetylated STAT3. Data were expressed as mean ± SD (n = 5). ## p < 0.01, ### p < 0.001, vs. sham rats; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. UUO rats.
Figure 7
Figure 7
The proposed pathway of TGF-β-induced pEMT in RTE cells and the inhibitory effect of rhein.
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