7-Ketolithocholic Acid Exerts Anti-Renal Fibrotic Effects Through FXR-Mediated Inhibition of TGF-β/Smad and Wnt/β-Catenin Pathways

Figures

• 

  Figure 1. 7-KLCA improves renal fibrosis in vitro. (A) In vitro cell viability test for HK-2 cells treated with 7-KLCA for 24 h (n = 6). CCK-8 assay results showed no significant decrease in cell viability when 7-KLCA concentration was ≤100 μM. Therefore, the non-cytotoxic doses of 2 μM, 5 μM, and 10 μM were selected for subsequent TGF-β-induced renal fibrosis experiments. (B) The protein expressions of Fibronectin, Collagen-I, Vimentin, and α-SMA in the HK-2 cells from different groups (n = 6). (C–E) Quantitative analysis of (B). Data represent the ratio of Fibronectin/Collagen-I/Vimentin band intensity to GAPDH (loading control), normalized to the TGF-β group. (F) In vitro cell viability test for NRK-49F cells treated with 7-KLCA for 24 h (n = 6). Consistent with the results in HK-2 cells, 7-KLCA did not affect cell viability at concentrations ≤100 μM. The non-toxic doses of 2 μM, 5 μM, and 10 μM were used in subsequent TGF-β-induced fibrosis experiments. (G) The protein expressions of Fibronectin, Collagen-I, Vimentin, and α-SMA in the NRK-49F cells from different groups (n = 6). (H–K) Quantitative analysis of (G). Data represent the ratio of Fibronectin/Collagen-I/Vimentin/α-SMA band…

• 

  Figure 2. 7-KLCA improves renal fibrosis in vivo. (A) UUO animal experimental programme protocol. (B) Picture of left kidneys of mice with different treatments (n = 3). (C) The renal index (mg/g) was calculated by dividing the wet renal weight by the bodyweight (n = 6). (D) Representative micrographs of H&E and Masson staining of the left kidneys of mice in different groups (scale bar: 50 μm; magnification: 200×). (E,F) The histogram of renal injury and renal interstitial fibrosis score according to the pathological results. (G) The expressions of Fibronectin, Collagen-I, Vimentin, and α-SMA in the kidneys of mice based on WB analysis (n = 6). (H–K) Quantitative analysis of (G). Data represent the ratio of Fibronectin/Collagen-I/Vimentin/α-SMA band intensity to GAPDH (loading control), normalized to the UUO group. (L) Ade animal experimental programme protocol. (M,N) The expression of SCR and BUN in sera of mice (n = 6). (O) Representative micrographs of H&E and Masson staining of the left kidneys of mice in different groups (scale bar: 50 μm; magnification: 200×). (P,Q) The histogram of renal injury and renal interstitial fibrosis score according to the pathological results. (R) The…

• 

  Figure 3. FXR expression was reduced in renal fibrosis. (A) Transcriptomic analysis of HK-2 cells, the volcano plot showing the differentially expressed genes between control and TGF-β groups (n = 3). (B) The mRNA levels of FXR based on the GEO database and the differential expression of FXR in the kidneys of 20 healthy individuals and 20 patients with IgA nephropathy compared using the Mann–Whitney Test. (C) The expressions of FXR in the kidneys of mice based on WB analysis (n = 6). (D) Quantitative analysis of (C). Data represent the ratio of FXR band intensity to GAPDH (loading control), normalized to the sham group. (E) Relative mRNA expression of FXR in the kidneys of UUO mice was assessed by qPCR. (F) The expressions of FXR in the kidneys of mice based on WB analysis (n = 6). (G) Quantitative analysis of (F). Data represent the ratio of FXR band intensity to GAPDH (loading control), normalized to the Control group. (H) Relative mRNA expression of FXR in the kidneys of Ade mice was assessed by qPCR. Results are mean ± SD of at least six independent experiments. *** p < 0.001.

• 

  Figure 4. 7-KLCA directly binds to FXR and upregulates its expression. (A) Results of molecular docking between 7-KLCA and FXR. (B) Quantitative parameter table for different docking modes. (C) WB detection results of cellular thermal shift analysis (n = 4). (D) Relative intensity quantification curve of the FXR band in (C). Data represent the ratio of FXR band intensity to GAPDH (loading control), normalized to the Control group (without 7-KLCA treatment). (E) The binding between 7-KLCA and FXR based on drug-affinity-responsive target stability assay (n = 4). (F) Quantitative analysis of (E). Data represent the ratio of FXR band intensity to GAPDH (loading control), normalized to the group without 7-KLCA pre-treatment. (G–I) Relative mRNA expression of FXR (G), SHP (H), and FGF15 (I) in the kidneys of UUO mice from sham (sham surgery), UUO (model), 7-KLCA-L (20 mg/kg), 7-KLCA-H (40 mg/kg), and Losartan (10 mg/kg) groups was assessed by qPCR. Data are presented as mean ± SD (n = 6), with statistical comparisons performed against the UUO model group. (J) The protein expressions of FXR in the HK-2 cells from different groups (n = 6). (K) Quantitative analysis of (J). Data represent the ratio…

• 

  Figure 5. Overexpression of FXR improves renal fibrosis and inhibits TGF-β/Smad signalling pathway. (A) The expression levels of FXR protein in HK-2 cells from different groups (n = 6). (B) Quantitative analysis of (A). Data represent the ratio of FXR band intensity to GAPDH (loading control), normalized to the Vector group. (C) The protein expressions of Fibronectin, Collagen-I, and Vimentin in the HK-2 cells from different groups (n = 6). (D–F) Quantitative analysis of (C). Data represent the ratio of Fibronectin/Collagen-I/Vimentin band intensity to GAPDH (loading control), normalized to the TGF-β group. (G) The protein expressions of TGF-β, Smad2, and Smad3 in the HK-2 cells from different groups (n = 6). (H–J) Quantitative analysis of (G). Data represent the ratio of TGF-β/Smad2/Smad3 band intensity to GAPDH (loading control), normalized to the TGF-β group. Results are mean ± SD of at least six independent experiments. *** p < 0.001.

• 

  Figure 6. 7-KLCA inhibits TGF-β/Smad and Wnt/β-catenin signalling pathway. (A) The expressions of TGF-β, Smad2, and Smad3 in the kidneys of mice based on WB analysis (n = 6). (B–D) Quantitative analysis of (A). Data represent the ratio of TGF-β/Smad2/Smad3 band intensity to GAPDH (loading control), normalized to the UUO group. (E) The expressions of Wnt5a/b, β-catenin, and Axin1 in the kidneys of mice based on WB analysis (n = 6). (F–H) Quantitative analysis of (E). Data represent the ratio of Wnt5a/b/β-catenin/Axin1 band intensity to GAPDH (loading control), normalized to the UUO group. (I) The expressions of TGF-β, Smad2, and Smad3 in the kidneys of mice based on WB analysis (n = 6). (J–L) Quantitative analysis of (I). Data represent the ratio of TGF-β/Smad2/Smad3 band intensity to GAPDH (loading control), normalized to the Ade group. (M) The expressions of Wnt5a/b, β-catenin, and Axin1 in the kidneys of mice based on WB analysis (n = 6). (N–P) Quantitative analysis of (M). Data represent the ratio of Wnt5a/b/β-catenin/Axin1 band intensity to GAPDH (loading control), normalized to the Ade group. Results are mean ± SD of at least six independent experiments. * p < 0.05, ** p < 0.01, *** p…

• 

  Figure 7. Schematic illustration of the anti-renal fibrotic effect of 7-KLCA.