Fig. 4: 1,5-AG as an agonist of TGR5 and knockdown of TGR5 abolishing the anti-fibrotic effect of 1,5-AG.

A Immunohistochemical micrographs of TGR5 in the kidney tissues of IgA patients (scale bar, 100 μm). B IHC scores of Fig. 4A (n = 3) **p =0.0021. C TGR5 protein expressions of kidney in UUO and adenine models. **p = 0.0054 for UUO model: Sham vs. UUO, p = 0.5034 for UUO model: Sham vs. 1, 5-AG, ^#p = 0.0237 for UUO model: UUO vs. 1, 5-AG; ***p = 0.0003 for adenine model: Control vs. Ade, *p = 0.0446 for adenine model: Control vs. 1, 5-AG, ^##p = 0.0034 for adenine model: Ade vs. 1, 5-AG. D TGR5, FN, Col-I and Vimentin expressions in primary mouse renal tubular cells (PRTC) after treatment with TGF-β (10 ng/mL) + high glucose (30 mM) and 1,5-AG (50 μM). E Quantitative analysis of Fig. 4D (n = 3). ***p = 0.0001 for TGR5: Control vs. Model, **p = 0.0028 for TGR5: Control vs. 1, 5-AG, ^#p = 0.0137 for TGR5: Model vs. 1, 5-AG; ***p  < 0.0001 for FN: Control vs. Model, ***p = 0.0006 for FN: Control vs. 1, 5-AG, ^##p = 0.0043 for FN: Model vs. 1, 5-AG; *p = 0.0149 for COL-1: Control vs. Model, p = 0.1713 for COL-1: Control vs. 1, 5-AG, ^#p = 0.0247 for COL-1: Model vs. 1, 5-AG, Comparison in COL-1 were performed with a two-tailed Student’s t test; **p = 0.0075 for VIMENTIN: Control vs. Model, *p = 0.0270 for VIMENTIN: Control vs. 1, 5-AG, ^#p = 0.0231 for VIMENTIN: Model vs. 1, 5-AG, Comparison in VIMENTIN were performed with a two-tailed Student’s t test. F The cAMP levels in adenine model. *p = 0.0130 for cAMP: Control vs. Ade, ^#p = 0.0376 for cAMP: Ade vs. 1, 5-AG. G The root mean square deviation (RMSD) of protein backbone atoms and 1,5-AG during the molecular dynamics (MD) simulation. H Molecular docking and the binding mode of 1,5-AG to TGR5 through the MD simulation. I Representative Western blot of Nrf2, Keap1, and HO-1. J Quantitative analysis of Fig. 4I (n = 3). **p = 0.0058 for NRF2: Control vs. Model, p = 0.9139 for NRF2: Control vs. 1, 5-AG, ^#p = 0.0107 for NRF2: Model vs. 1, 5-AG; ***p  < 0.0001 for KEAP1: Control vs. Model, **p = 0.0015 for KEAP1: Control vs. 1, 5-AG, ^##p = 0.0048 for KEAP1: Model vs. 1, 5-AG; **p = 0.0029 for HO-1: Control vs. Model, *p = 0.0261 for HO-1: Control vs. 1, 5-AG, ^##p = 0.0097 for HO-1: Model vs. 1, 5-AG, Comparison in HO-1 were performed with a two-tailed Student’s t test. K FN, Col-I and Vimentin expressions in PRTC after treatment with siRNA against TGR5 or negative control. L Quantitative analysis of panel K (n = 3). ***p < 0.0001 for FN; **p = 0.0027 for COL-1; *p = 0.0117 for VIMENTIN. M The effect of SBI-115 on anti-fibrotic effect of 1,5-AG in PRTC. N Quantitative analysis of panel M (n = 3). **p = 0.0023 for FN; **p = 0.0036 for COL-1; ***p = 0.0002 for VIMENTIN. Data are presented as mean ± SD. Comparison in B were performed with a two-tailed Student’s t test. Comparisons in C, E, F, J, L and N were compared using One-Way ANOVA followed by Sidak’s multiple comparisons test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (compared with sham or control group), ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 (compared with model group). Individual data points are independent biological replicates unless otherwise stated.